Matrix-free osteogenic devices, implants and methods of use thereof

ABSTRACT

Provided herein are methods for inducing bone formation in a mammal sufficient to fill a defect defining a void, wherein osteogenic protein is provided alone or dispersed in a biocompatible non-rigid, amorphous carrier having no defined surfaces. The methods and devices provide injectable formulations for filling critical size defects, as well as for accelerating the rate and enhancing the quality of bone formation in non-critical size defects.

PRIORITY APPLICATION DATA

This application claims the benefit of prior applications, U.S. Ser. No.60/037,327 filed Feb. 7, 1997, and U.S. Ser. No. 60/047,909, filed May29, 1997, the entire contents of both of which are incorporated byreference herein.

FIELD OF THE INVENTION

The invention disclosed herein relates to materials and methods forrepairing bone defects using osteogenic proteins.

BACKGROUND OF THE INVENTION

A class of proteins now have been identified that are competent to actas true chondrogenic tissue morphogens, able, on their own, to inducethe proliferation and differentiation of progenitor cells intofunctional bone, cartilage, tendon, and/or ligamentous tissue. Theseproteins, referred to herein as “osteogenic proteins” or “morphogenicproteins” or “morphogens,” includes members of the family of bonemorphogenic proteins (BMPs) which were initially identified by theirability to induce ectopic, endochondral bone morphogenesis. Theosteogenic proteins generally are classified in the art as a subgroup ofthe TGF-β superfamily of growth factors (Hogan (1996) Genes &Development 10:1580-1594). Members of the morphogen family of proteinsinclude the mammalian osteogenic protein-1 (OP-1, also known as BMP-7,and the Drosophila homolog 60A), osteogenic protein-2 (OP-2, also knownas BMP-8), osteogenic protein-3 (OP-3), BMP-2 (also known as BMP-2A orCBMP-2A, and the Drosophila homolog DPP), BMP-3, BMP-4 (also known asBMP-2B or CBMP-2B), BMP-5, BMP-6 and its murine homolog Vgr-1, BMP-9,BMP-10, BMP-11, BMP-12, GDF3 (also known as Vgr2), GDF8, GDF9, GDF10,GDF11, GDF12, BMP-13, BMP-14, BMP-15, GDF-5 (also known as CDMP-1 orMP52), GDF-6 (also known as CDMP-2), GDF-7 (also known as CDMP-3), theXenopus homolog Vgl and NODAL, UNIVIN, SCREW, ADMP, and NEURAL. Membersof this family encode secreted polypeptide chains sharing commonstructural features, including processing from a precursor “pro-form” toyield a mature polypeptide chain competent to dimerize, and containing acarboxy terminal active domain of approximately 97-106 amino acids. Allmembers share a conserved pattern of cysteines in this domain and theactive form of these proteins can be either a disulfide-bonded homodimerof a single family member, or a heterodimer of two different members(see, e.g., Massague (1990) Annu. Rev. Cell Biol. 6:597; Sampath, et al.(1990) J. Biol. Chem. 265:13198). See also, U.S. Pat. No. 5,011,691;U.S. Pat. No. 5,266,683, Ozkaynak et al. (1990) EMBO J. 9:2085-2093,Wharton et al. (1991) PNAS 88:9214-9218), (Ozkaynak (1992) J. Biol.Chem. 267:25220-25227 and U.S. Pat. No. 5,266,683); (Celeste et al.(1991) PNAS 87:9843-9847); (Lyons et al. (1989) PNAS 86:4554-4558).These disclosures describe the amino acid and DNA sequences, as well asthe chemical and physical characteristics of these osteogenic proteins.See also Wozney et al. (1988) Science 242:1528-1534); BMP 9 (WO93/00432,published Jan. 7, 1993); DPP (Padgett et al. (1987) Nature 325:81-84;and Vgl (Weeks (1987) Cell 51:861-867).

Thus, true osteogenic proteins capable of inducing the above-describedcascade of morphogenic events that result in endochondral bone formationhave now been identified, isolated, and cloned. Whethernaturally-occurring or synthetically prepared, these osteogenic factors,when implanted in a mammal in association with a conventional matrix orsubstrate that allows the attachment, proliferation and differentiationof migratory progenitor cells, have been shown to induce recruitment ofaccessible progenitor cells and stimulate their proliferation, therebyinducing differentiation into chondrocytes and osteoblasts, and furtherinducing differentiation of intermediate cartilage, vascularization,bone formation, remodeling, and finally marrow differentiation.Furthermore, numerous practitioners have demonstrated the ability ofthese osteogenic proteins, when admixed with either naturally-sourcedmatrix materials such as collagen or synthetically-prepared polymericmatrix materials, to induce bone formation, including endochondral boneformation under conditions where true replacement bone otherwise wouldnot occur. For example, when combined with a matrix material, theseosteogenic proteins induce formation of new bone in: large segmentalbone defects, spinal fusions, and fractures. Without exception, each ofthe above-referenced disclosures describes implantation or delivery ofthe osteogenic protein at the defect site by packing, filling, and/orwrapping the defect site with an admixture of osteogenic protein andmatrix, with the relative volume and surface area of matrix beingsignificant. In the case of non-union defects which do not healspontaneously, it has heretofore been conventional practice to implantvolumes of matrix-osteogenic factor admixtures at the defect site, thevolumes being sufficient to fill the defect in order to provide a3-dimensional scaffold for subsequent new bone formation. While standardbone fractures, can heal spontaneously and without treatment, to theextent the art has contemplated treating fractures with osteogenicproteins, it has been the practice in the art to provide the osteogenicprotein together with a matrix locally to a defect site to promotehealing.

While implanting a volume of matrix may be conventional wisdom,particularly in the case of non-healing non-union defects, clinicalconsequences may develop in certain patients as a result of thispractice. For example, patients undergoing repeated constructions ordefect repairs, or wherein the matrix volume is large, can developadverse immunologic reactions to matrices derived from collagen.Collagen matrices can be purified, but residual levels of contaminantscan remain which is strongly allergenic for certain patients.Alternatively, demineralized autogenic, allogenic or xenogenic bonematrix can be used in place of collagen. Such a matrix is mechanicallysuperior to collagen and can obviate adverse immune reactions in somecases, but proper preparation is expensive, time consuming andavailability of reliable sources for bone may be limited. Suchnaturally-sourced matrices can be replaced with inert materials such asplastic, but plastic is not a suitable substitute since it does notresorb and is limited to applications requiring simple geometricconfigurations. To date, biodegradable polymers and copolymers have alsobeen used as matrices admixed with osteogenic proteins for repair ofnon-union defects. While such matrices may overcome some of theabove-described insufficiencies, use of these matrices stillnecessitates determination and control of features such as polymerchemistry, particle size, biocompatability and other particularscritical for operability.

In addition, individuals who, due to an acquired or congenitalcondition, have a reduced ability to heal bone fractures or otherdefects that normally undergo spontaneous repair would benefit frommethods and injectable compositions that can enhance bone and/orcartilage repair without requiring a surgical procedure. Finally, aninjectable formulation also provides means for repairing osteochondralor chondral defects without requiring a surgical procedure.

Needs remain for devices, implants and methods of repairing bone defectswhich do not rely on a matrix component. Particular needs remain fordevices, implants and methods which permit delivery of bone-inducingamounts of osteogenic proteins without concomitant delivery ofspace-filling matrix materials which can compromise the recipient and/orfail to be biomechanically and torsionally ideal. Needs also remain forproviding methods and devices, particularly injectable devices that canaccelerate the rate and enhance the quality of new bone formation.

Accordingly, it is an object of the instant invention to providedevices, implants and methods of use thereof for repairing bone defects,cartilage defects and/or osteochondral defects which obviate the needfor an admixture of osteogenic protein with matrix. The instantinvention provides matrix-free osteogenic devices, implants and methodsof use thereof for repairing non-healing non-union defects, as well asfor promoting enhanced bone formation for spinal fusions and bonefractures, and for promoting articular cartilage repair in chondral orosteochondral defects. These and other objects, along with advantagesand features of the invention disclosed herein, will be apparent fromthe description, drawings and claims that follow.

SUMMARY OF THE INVENTION

The present invention is based on the discovery that an osteogenic orbone morphogenic protein such as OP-1, alone or when admixed with asuitable carrier and not with a conventional matrix material, can induceendochondral bone formation sufficient to repair critical-sized,segmental bone defects. Thus this discovery overcomes theabove-described problems associated with conventional materials andmethods for repairing bone defects because it permits elimination ofmatrix material. Furthermore, in view of existing orthopedic andreconstructive practices, this discovery is unexpected and contravenesthe art's current understanding of the bone repair/formation processes.

As disclosed herein, it is now appreciated that an osteogenic proteincan be admixed with a carrier as defined herein to form a matrix-freedevice which, when provided to a mammal, is effective to promote repairof non-union bone defects, fractures and fusions. As disclosed herein,methods and devices are provided for inducing new bone formation at alocal defect site without the need for also providing athree-dimensional structural component at the defect site. Ascontemplated herein, a “matrix-free” osteogenic device is a devicedevoid of matrix at the time it is provided to a recipient. It isunderstood that the term “matrix” means a structural component orsubstrate having a three-dimensional form and upon which certaincellular events involved in endochondral bone morphogenesis will occur;a matrix acts as a temporary scaffolding structure for infiltratingcells having interstices for attachment, proliferation anddifferentiation of such cells.

The invention provides, in one aspect therefore, a novel method forinducing bone formation in a mammal sufficient to repair a defect. Oneembodiment comprises the step of providing a matrix-free osteogenicdevice to a defect locus defining a void. The matrix-free device may becomposed of osteogenic protein alone, or it may be composed ofosteogenic protein in admixture with a biocompatible, amorphousnon-rigid carrier having no defined surfaces. This method induces newbone formation which fills the defect locus, thereby repairing thedefect. As contemplated herein, the method comprises providing amatrix-free osteogenic device to a defect locus, wherein the device isprovided in a volume insufficient to fill the void at the defect locus.In certain embodiments, the void comprises a volume incapable ofendogenous or spontaneous repair. Examples of defects suitable forrepair by the instant method include, but are not limited to,critical-sized segmental defects and non-union fractures.

In another embodiment, the invention provides methods and compositionsfor enhancing fracture repair by providing the matrix-free osteogenicdevices described herein to a fracture defect site. The ability of thedevices described herein to substantially enhance fracture repair,including accelerating the rate and enhancing the quality of newlyformed bone, has implications for improving bone healing in compromisedindividuals such as diabetics, smokers, obese individuals and otherswho, due to an acquired or congenital condition have a reduced capacityto heal bone fractures, including individuals with impaired blood flowto their extremities.

In another aspect, the invention provides an implant for inducing boneformation in a mammal sufficient to repair a defect. One preferredimplant comprises a matrix-free osteogenic device disposed at a defectlocus defining a void. Practice of the above-described method, i.e.,providing an osteogenic device devoid of scaffolding structure to amammal at a defect locus, results in an implant competent to induce newbone formation sufficient to promote repair of non-union bone defects,fractures and fusions. Upon disposition of the osteogenic device at thedefect locus, the implant so formed has insufficient volume to fill thedefect void.

In yet another aspect, the present invention provides a matrix-freeosteogenic device for inducing bone formation in a mammal. Ascontemplated herein, a preferred osteogenic device comprises anosteogenically-active protein dispersed in a suitable carrier. Preferredosteogenic proteins, include but are not limited to, OP-1, OP-2, BMP-2,BMP-4, BMP-5, and BMP-6 (see below). As disclosed herein, preferredcarriers are biocompatible, nonrigid and amorphous, having no definedsurfaces or three-dimensional structural features. Thus, the devices ofthe instant invention lack scaffolding structure and are substantiallyfree of matrix when administered to a mammal. Examples of preferredcarriers include, but are not limited to, poloxamers andalkylcelluloses. As discussed above, the method of the instant inventioninvolves providing such a device to a defect locus such that the volumeof the device is insufficient to fill the void volume at the defectlocus.

The methods, implants and devices of the invention also are competent toinduce and promote or enhance repair of chondral or osteochondraldefects. As a result of this discovery means now are available forpromoting bone and/or cartilage repair without requiring a surgicalprocedure. Particularly as a method for enhancing bone fracture repair,it is contemplated that a suitable formulation can be injected to afracture site at the time the fracture is set so as to accelerate therate and enhance the quality of new bone formation.

The device of the instant invention can have a variety ofconfigurations. The nature of the device will be dependent upon the typeof carrier in which the osteogenic protein is dispersed. For example,one preferred embodiment can have a paste-like or putty-likeconfiguration; such a device can result from dispersing osteogenicprotein in a gel-like carrier such as a Pluronic™ carrier or analkylcellulose such as carboxymethyl cellulose which is then wetted witha suitable wetting agent such as, for example, a saline solution.Another preferred embodiment can have a dry powder configuration; such adevice results from first dispersing osteogenic protein in a liquidcarrier such as water with or without excipient, followed bylyophilization. A third formulation is a solution, such as by combiningthe protein together with an acidic buffered solution, e.g., pH 4.0-4.5,for example an acetate or citrate buffer. Still another formulation is asuspension formed by disbursing osteogenic protein in a physiologicallybuffered solution, such as phosphate buffered saline (PBS). Dependingupon the configuration of the device, providing it to a defect locus canbe accomplished by a variety of delivery processes. For example, a pastecan be extruded as a bead which lays along one surface of the defectlocus. Alternatively, a viscous liquid can be brushed or painted alongone or more surfaces of the defect locus or injected through a widegauge needle. Less viscous fluids can be injected through a fine gaugeneedle. Other configurations and modes of delivery are contemplated anddiscussed below in more detail.

Generally, the proteins of the invention are dimeric proteins thatinduce endochondral bone morphogenesis. Osteogenic proteins comprise apair of polypeptides that, when folded, adopt a configuration sufficientfor the resulting dimeric protein to elicit a morphogenic response. Thatis, osteogenic proteins generally induce all of the following biologicalfunctions in a morphogenically permissive environment: stimulatingproliferation of progenitor cells; stimulating the differentiation ofprogenitor cells; stimulating the proliferation of differentiated cells;and supporting the growth and maintenance of differentiated cells.Progenitor cells are uncommitted cells that are competent todifferentiate into one or more specific types of differentiated cells,depending on their genomic repertoire and the tissue specificity of thepermissive environment in which morphogenesis is induced. In the instantinvention, osteogenic proteins can induce the morphogenic cascade whichtypifies endochondral bone formation.

As used herein, the term “morphogen”, “bone morphogen”, “bonemorphogenic protein”, “BMP”, “osteogenic protein” and “osteogenicfactor” embraces the class of proteins typified by human osteogenicprotein 1 (hOP-1). Nucleotide and amino acid sequences for hOP-1 areprovided in Seq. ID Nos. 1 and 2, respectively. For ease of description,hOP-1 is recited herein below as a representative osteogenic protein. Itwill be appreciated by the artisan of ordinary skill in the art,however, that OP-1 merely is representative of the TGF-β subclass oftrue tissue morphogenes competent to act as osteogenic proteins, and isnot intended to limit the description. Other known, and useful proteinsinclude, BMP-2, BMP-3, BMP-3b, BMP-4, BMP-5, BMP-6, BMP-8, BMP-9,BMP-10, BMP-11, BMP-12, BMP-13, BMP-15, GDF-1, GDF-2, GDF-3, GDF-5,GDF-6, GDF-7, GDF-8, GDF-9, GDF-10, GDF-11, GDF-12, NODAL, UNIVIN,SCREW, ADMP, NURAL and osteogenically active amino acid variantsthereof. In one preferred embodiment, the proteins useful in theinvention include biologically active species variants of any of theseproteins, including conservative amino acid sequence variants, proteinsencoded by degenerate nucleotide sequence variants, and osteogenicallyactive proteins sharing the conserved seven cysteine skeleton as definedherein and encoded by a DNA sequence competent to hybridize to a DNAsequence encoding an osteogenic protein disclosed herein. In stillanother embodiment, useful osteogenic proteins include those sharing theconserved seven cysteine domain and sharing at least 70% amino acidsequence homology (similarity) within the C-terminal active domain, asdefined herein.

In still another embodiment, the osteogenic proteins of the inventioncan be defined as osteogenically active proteins having any one of thegeneric sequences defined herein, including OPX and Generic Sequences 7(SEQ ID NO:4) and 8 (SEQ ID NO:5) or Generic Sequences 9 (SEQ ID NO:8)and 10 (SEQ ID NO:7). OPX accommodates the homologies between thevarious species of the osteogenic OP1 and OP2 proteins, and is describedby the amino acid sequence presented herein below and in Seq. ID No. 3.Generic sequence 9 (SEQ ID NO:8) is a 102 amino acid sequence containingthe six cysteine skeleton defined by hOP1 (residues 330-431 of Seq. IDNo. 2) and wherein the remaining residues accommodate the homologies ofOP1, OP2, OP3, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-8, BMP-9, BMP-10,BMP-11, BMP-15, GDF-1, GDF-3, GDF-5, GDF-6, GDF-7, GDF-8, GDF-9, GDF-10,GDF-11, UNIVIN, NODAL, DORSALIN, NURAL, SCREW and ADMP. That is, each ofthe non-cysteine residues is independently selected from thecorresponding residue in this recited group of proteins. Genericsequence 10 (SEQ ID NO:7) is a 97 amino acid sequence containing theseven cysteine skeleton defined by hOPI (335-431 Seq. ID No. 2) andwherein the remaining residues accommodate the homologies of theabove-recited protein group.

As contemplated herein, this family of osteogenic proteins includeslonger forms of a given protein, as well as phylogenetic, e.g., speciesand allelic variants and biosynthetic mutants, including C-terminaladdition and deletion mutants and variants, such as those which mayalter the conserved C-terminal cysteine skeleton, provided that thealteration still allows the protein to form a dimeric species having aconformation capable of inducing bone formation in a mammal whenimplanted in the mammal. In addition, the osteogenic proteins useful inthis invention may include forms having varying glycosylation patternsand varying N-termini, may be naturally occurring or biosyntheticallyderived, and may be produced by expression of recombinant DNA inprocaryotic or eucaryotic host cells. The proteins are active as asingle species (e.g., as homodimers), or combined as a mixed species,including heterodimers.

The methods and implants of the invention do not require a carrier forthe osteogenic protein to induce bone formation sufficient to fill acritical size bone defect or to enhance fracture repair in an animal.When the protein is provided in association with a carrier in thepractice of the invention, the carrier must lack a scaffoldingstructure, as stated above. When a preferred carrier is admixed with anosteogenic protein, a device is formed which is substantially free ofmatrix as defined herein. “Substantially free of matrix” is understoodto mean that, the carrier-containing device as formulated prior toadministration, does not contain a substrate competent to act as ascaffold per se. That is, the device contains no substrate which hasbeen introduced from an exogenous source and is competent to act as ascaffold. Stated another way, prior to delivery, the carrier isrecognized, by virtue of its chemical nature, to be unable to contributea scaffolding structure to the device. By definition, preferred carriersare biocompatible, non-rigid and amorphous, having no defined surfaces.As used herein, “non-rigid” means a carrier formulation that is lax orpliant or otherwise is substantially incapable of providing or forming athree-dimensional structure having one or more defined surfaces. As usedherein, “amorphous” means lacking a definite three-dimensional form, orspecific shape, that is, having no particular shape or form, or havingan indeterminate shape or form. Preferred carriers are alsobiocompatible, non-particulate, adherent to bone, cartilage and/ormuscle, and inert. In certain embodiments, water-soluble carriers arepreferable. Additionally, preferred carriers do not contributesignificant volume to a device of the instant invention. That is, apreferred carrier permits dispersal of an osteogenic protein such thatthe final volume of the resulting device is less than the volume of thevoid at the defect locus. As discussed below, a preferred carrier can bea gel, an aqueous solution, a suspension or a viscous liquid. Forexample, particularly preferred carriers can include, withoutlimitation, poloxamers alkylcelluloses, acetate buffers, physiologicalsaline solutions, lactose, mannitol and/or other sugars. Alternatively,osteogenic proteins can be provided alone to a defect site.

In summary, the methods, implants and devices of the present inventioncan be used to induce endochondral and intramembranous bone formationsufficient to repair bone defects which do not heal spontaneously, aswell as to promote and enhance the rate and/or quality of new boneformation, particularly in the repair of fractures and fusions,including spinal fusions. The methods, implants and devices also arecompetent to induce repair of osteochondral and/or subchondral defects.That is, the methods, implants and devices are competent to induceformation of new bone and the overlying surface cartilage. The presentinvention is particularly suitable for use in collagen- ormatrix-allergenic recipients. It is also particularly suitable for usein patients requiring repetitive reconstructive surgeries, as well ascancer patients as an alternative to reconstructive procedures usingmetal joints. The present invention also is useful for individuals whoseability to undergo spontaneous bone repair is compromised, such asdiabetics, smokers, obese individuals, immune-compromised individuals,and any individuals have reduced blood flow to their extremities. Otherapplications include, but are not limited to, prosthetic repair, spinalfusion, scoliosis, cranial/facial repair, and massive allograft repair.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In order to more clearly and concisely describe the subject matter ofthe claimed invention, the following definitions are intended to provideguidance as to the meaning of specific terms used in the followingwritten description and appended claims.

“Bone formation” means formation of endochondral bone or formation ofintramembranous bone. In humans, bone formation begins during the first6-8 weeks of fetal development. Progenitor stem cells of mesenchymalorigin migrate to predetermined sites, where they either: (a) condense,proliferate, and differentiate into bone-forming cells (osteoblasts), aprocess observed in the skull and referred to as “intramembranous boneformation;” or, (b) condense, proliferate and differentiate intocartilage-forming cells (chondroblasts) as intermediates, which aresubsequently replaced with bone-forming cells. More specifically,mesenchymal stem cells differentiate into chondrocytes. The chondrocytesthen become calcified, undergo hypertrophy and are replaced by newlyformed bone made by differentiated osteoblasts which now are present atthe locus. Subsequently, the mineralized bone is extensively remodeled,thereafter becoming occupied by an ossicle filled with functionalbone-marrow elements. This process is observed in long bones andreferred to as “endochondral bone formation.” In postfetal life, bonehas the capacity to repair itself upon injury by mimicking the cellularprocess of embryonic endochondral bone development. That is, mesenchymalprogenitor stem cells from the bone-marrow, periosteum, and muscle canbe induced to migrate to the defect site and begin the cascade of eventsdescribed above. There, they accumulate, proliferate, and differentiateinto cartilage which is subsequently replaced with newly formed bone.

“Defect” or “defect locus” as contemplated herein defines a void whichis a bony structural disruption requiring repair. The defect further candefine an osteochondral defect, including both a structural disruptionof the bone and overlying cartilage. “Void” is understood to mean athree-dimensional defect such as, for example, a gap, cavity, hole orother substantial disruption in the structural integrity of a bone orjoint. A defect can be the result of accident, disease, surgicalmanipulation and/or prosthetic failure. In certain embodiments, thedefect locus is a void having a volume incapable of endogenous orspontaneous repair. Such defects are also called critical-sizedsegmental defects. The art recognizes such defects to be approximately3-4 cm, at least greater than 2.5 cm, gap incapable of spontaneousrepair. In other embodiments, the defect locus is a non-criticalsegmental defect approximately at least 0.5 cm but not more thanapproximately 2.5 cm. Generally, these are capable of some spontaneousrepair, albeit biomechanically inferior to that made possible bypractice of the instant innovation. In certain other embodiments, thedefect is an osteochondral defect such as an osteochondral plug. Otherdefects susceptible to repair using the instant invention include, butare not limited to, non-union fractures; bone cavities; tumor resection;fresh fractures; cranial/facial abnormalities; spinal fusions, as wellas those resulting from diseases such as cancer, arthritis, includingosteoarthritis, and other bone degenerative disorders. “Repair” isintended to mean induction of new bone formation which is sufficient tofill the void at the defect locus, but “repair” does not mean orotherwise necessitate a process of complete healing or a treatment whichis 100% effective at restoring a defect to its pre-defectphysiological/structural state.

“Matrix” is understood in the art to mean an osteoconductive substratehaving a scaffolding structure on which infiltrating cells can attach,proliferate and participate in the morphogenic process culminating inbone formation. In certain embodiments, matrix can be particulate andporous, with porosity being a feature critical to its effectiveness ininducing bone formation, particularly endochondral bone formation. Asdescribed earlier, a matrix is understood to provide certain structuralcomponents to the conventional osteogenic device (i.e., heretoforecomprising a porous, particulate matrix component such as collagen,demineralized bone or synthetic polymers), thereby acting as a temporaryand resorbable scaffolding structure for infiltrating cells havinginterstices for attachment, proliferation and differentiation of suchcells. Accordingly, the term “matrix-free osteogenic device” or anosteogenic device which is “substantially free of matrix” contemplates adevice which is devoid of an art-recognized matrix at the time it isprovided to a recipient. Moreover, substantially free of matrix isunderstood to mean that, when a device is provided to a defect locus, nosubstrate competent to act as a scaffold per se is introduced from anexogenous source. Matrix-free or substantially free of matrix is notintended to exclude endogenous matrix which is induced or formedfollowing delivery of the devices and/or implants disclosed herein to adefect locus. Thus the present invention further contemplates a methodof inducing endogenous matrix formation by providing to a defect locusthe matrix-free devices or implants disclosed herein.

“Osteogenic device” is understood to mean a composition comprisingosteogenic protein dispersed in a biocompatible, non-rigid amorphouscarrier having no defined surfaces. Osteogenic devices of the presentinvention are competent to induce bone formation sufficient to fill adefect locus defining a void. Osteogenic devices are matrix-free whenprovided to the defect locus and are delivered to the defect locus in avolume insufficient to fill the void defined by the defect locus. Adevice can have any suitable configuration, such as liquid, powder,paste, or gel, to name but a few. Preferred properties of osteogenicdevices suitable for use with the method of the instant inventioninclude, but are not limited to: adherent to bone, cartilage and/ormuscle; and, effective to provide at least a local source of osteogenicprotein at the defect locus, even if transient. As contemplated herein,providing a local source of protein includes both retaining protein atthe defect locus as well as controlled release of protein at the defectlocus. All that is required by the present invention is that theosteogenic device be effective to deliver osteogenic protein at aconcentration sufficient to induce bone formation that fills thethree-dimensional defect defining the void requiring repair. In additionto osteogenic proteins, various growth factors, hormones, enzymes,therapeutic compositions, antibiotics, or other bioactive agents canalso be contained within an osteogenic device. Thus, various knowngrowth factors such as EGF, PDGF, IGF, FGF, TGF-α, and TGF-β can becombined with an osteogenic device and be delivered to the defect locus.An osteogenic device can also be used to deliver chemotherapeuticagents, insulin, enzymes, enzyme inhibitors and/orchemoattractant/chemotactic factors.

“Osteogenic protein” or bone morphogenic protein is generally understoodto mean a protein which can induce the full cascade of morphogenicevents culminating in endochondral bone formation. As describedelsewhere herein, the class of proteins is typified by human osteogenicprotein (hOP 1). Other osteogenic proteins useful in the practice of theinvention include osteogenically active forms of OP1, OP2, OP3, BMP2,BMP3, BMP4, BMP5, BMP6, BMP9, DPP, Vgl, Vgr, 60A protein, GDF-1, GDF-3,GDF-5, 6, 7, BMP10, BMP11, BMP13, BMP15, UNIVIN, NODAL, SCREW, ADMP orNURAL and amino acid sequence variants thereof. In one currentlypreferred embodiment, osteogenic protein include any one of: OP1, OP2,OP3, BMP2, BMP4, BMP5, BMP6, BMP9, and amino acid sequence variants andhomologs thereof, including species homologs, thereof. Particularlypreferred osteogenic proteins are those comprising an amino acidsequence having at least 70% homology with the C-terminal 102-106 aminoacids, defining the conserved seven cystein domain, of human OP-1, BMP2,and related proteins. Certain preferred embodiments of the instantinvention comprise the osteogenic protein, OP-1. Certain other preferredembodiments comprise mature OP-1 solubilized in a physiological salinesolution. As further described elsewhere herein, the osteogenic proteinssuitable for use with Applicants' invention can be identified by meansof routine experimentation using the art-recognized bioassay describedby Reddi and Sampath. “Amino acid sequence homology” is understoodherein to mean amino acid sequence similarity. Homologous sequencesshare identical or similar amino acid residues, where similar residuesare conservative substitutions for, or allowed point mutations of,corresponding amino acid residues in an aligned reference sequence.Thus, a candidate polypeptide sequence that shares 70% amino acidhomology with a reference sequence is one in which any 70% of thealigned residues are either identical to or are conservativesubstitutions of the corresponding residues in a reference sequence.Examples of conservative variations include the substitution of onehydrophobic residue such as isoleucine, valine, leucine or methioninefor another, or the substitution of one polar residue for another, suchas the substitution of arginine for lysine, glutamic for aspartic acids,or glutamine for asparagine, and the like. The term “conservativevariation” also includes the use of a substituted amino acid in place ofan unsubstituted parent amino acid provided that antibodies raised tothe substituted polypeptide also immunoreact with the unsubstitutedpolypeptide.

Proteins useful in this invention include eukaryotic proteins identifiedas osteogenic proteins (see U.S. Pat. No. 5,011,691, incorporated hereinby reference), such as the OP-1, OP-2, OP-3 and CBMP-2 proteins, as wellas amino acid sequence-related proteins such as DPP (from Drosophila),Vgl (from Xenopus), Vgr-1 (from mouse), GDF-1 (from humans, see Lee(1991), PNAS 88:4250-4254), 60A (from Drosophila, see Wharton et al.(1991) PNAS 88:9214-9218), dorsalin-1 (from chick, see Basler et al.(1993) Cell 73:687-702 and GenBank accession number L12032) and GDF-5(from mouse, see Storm et al. (1994) Nature 368:639-643). BMP-3 is alsopreferred. Additional useful proteins include biosynthetic morphogenicconstructs disclosed in U.S. Pat. No. 5,011,691, e.g., COP-1, 3-5, 7 and16, as well as other proteins known in the art. Still other proteinsinclude osteogenically active forms of BMP-3b (see Takao, et al.,(1996), Biochem. Biophys. Res. Comm. 219: 656-662. BMP-9 (seeWO95/33830), BMP-15 (see WO96/35710), BMP-12 (see WO95/16035), CDMP-1(see WO 94/12814), CDMP-2 (see WO94/12814), BMP-10 (see WO94/26893),GDF-1 (see WO92/00382), GDF-10 (see WO95/10539), GDF-3 (see WO94/15965)and GDF-7 (WO95/01802).

Still other useful proteins include proteins encoded by DNAs competentto bybridize to a DNA encoding an osteogenic protein as describedherein, and related analogs, homologs, muteins and the like (see below).

“Carrier” as used herein means a biocompatible, non-rigid, amorphousmaterial having no defined surfaces suitable for use with the devices,implants and methods of the present invention. As earlier stated,“non-rigid” means a carrier formulation that is lax or plaint orotherwise is substantially incapable of providing or forming athree-dimensional structure having one or more defined surfaces. As usedherein, “amorphous” means lacking a definite three-dimensional form, orspecific shape, that is, having no particular shape or form, or havingan indeterminate shape or form. Suitable carriers also arenon-particulate and are non-porous, i.e., are pore-less. Carrierssuitable for use in the instant invention lack a three-dimensionalscaffolding structure and are substantially matrix-free. Thus,“substantially free of matrix” is also understood to mean that, when acarrier-containing device is provided to a defect locus, no substratecompetent to act as a scaffold per se is introduced from any exogenoussource, including the carrier. Prior to delivery to and implantation inthe recipient, the carrier is recognized by virtue of its chemicalnature to be unable to contribute a three-dimensional scaffoldingstructure to the device. Preferred carriers are adherent, at leasttransiently, to tissues such as bone, cartilage and/or muscles. Certainpreferred carriers are water-soluble, viscous, and/or inert.Additionally, preferred carriers do not contribute significant volume toa device. Currently preferred carriers include, without limitationalkylcelluloses, poloxamers, gelatins, polyethylene glycols, dextrins,vegetable oils and sugars. Particularly preferred carriers currentlyinclude but are not limited to Pluronic F127™ poloxamercarboxymethylcelluloses, lactose, mannitol and sesame oil. Otherpreferred carriers include acetate buffer (20 mM, pH 4.5), physiologicalsaline (PBS), and citrate buffer. In the case of devices comprisingcarriers such as acetate, Poloxamers and PBS, administration byinjection can result in precipitation of certain osteogenic proteins atthe administration site.

“Implant” as contemplated herein comprises osteogenic protein dispersedin a biocompatible, nonrigid amorphous carrier having no definedsurfaces disposed at a defect locus defining a void. That is, theimplant of the present invention is contemplated to comprise the defectlocus per se into/onto which the device of the present invention hasbeen delivered/deposited. It is further contemplated that, at the timeof delivery of a device, an implant lacks scaffolding structure and issubstantially matrix-free. Implants resulting from practice of theinstant method are competent to induce bone formation in a defect locusin a mammal sufficient to fill the defect with newly formed bone withoutalso requiring inclusion of a matrix or scaffolding structure, at thetime of delivery of a device, sufficient to substantially fill the voidthereby structurally defining the defect size and shape.

The means for making and using the methods, implants and devices of theinvention, as well as other material aspects concerning their nature andutility, including how to make and how to use the subject matterclaimed, will be further understood from the following, whichconstitutes the best mode currently contemplated for practicing theinvention. It will be appreciated that the invention is not limited tosuch exemplary work or to the specific details set forth in theseexamples.

I. PROTEIN CONSIDERATIONS A. Biochemical, Structural and FunctionalProperties of Bone Morphogenic Proteins

Naturally occurring proteins identified and/or appreciated herein to beosteogenic or bone morphogenic proteins form a distinct subgroup withinthe loose evolutionary grouping of sequence-related proteins known asthe TGF-β superfamily or supergene family. The naturally occurring bonemorphogens share substantial amino acid sequence homology in theirC-terminal regions (domains). Typically, the above-mentioned naturallyoccurring osteogenic proteins are translated as a precursor, having anN-terminal signal peptide sequence, typically less than about 30residues, followed by a “pro” domain that is cleaved to yield the matureC-terminal domain. The signal peptide is cleaved rapidly upontranslation, at a cleavage site that can be predicted in a givensequence using the method of Von Heijne (1986) Nucleic Acids Research14:4683-4691. The pro domain typically is about three times larger thanthe fully processed mature C-terminal domain. Herein, the “pro” form ofa morphogen refers to a morphogen comprising a folded pair ofpolypeptides each comprising the pro and mature domains of a morphogenpolypeptide. Typically, the pro form of a morphogen is more soluble thanthe mature form under physiological conditions. The pro form appears tobe the primary form secreted from cultured mammalian cells.

In preferred embodiments, the pair of morphogenic polypeptides haveamino acid sequences each comprising a sequence that shares a definedrelationship with an amino acid sequence of a reference morphogen.Herein, preferred osteogenic polypeptides share a defined relationshipwith a sequence present in osteogenically active human OP-1, SEQ ID NO:2. However, any one or more of the naturally occurring or biosyntheticsequences disclosed herein similarly could be used as a referencesequence. Preferred osteogenic polypeptides share a defined relationshipwith at least the C-terminal six cysteine domain of human OP-1, residues335-431 of SEQ ID NO: 2. Preferably, osteogenic polypeptides share adefined relationship with at least the C-terminal seven cysteine domainof human OP-1, residues 330-431 of SEQ ID NO: 2. That is, preferredpolypeptides in a dimeric protein with bone morphogenic activity eachcomprise a sequence that corresponds to a reference sequence or isfunctionally equivalent thereto.

Functionally equivalent sequences include functionally equivalentarrangements of cysteine residues disposed within the referencesequence, including amino acid insertions or deletions which alter thelinear arrangement of these cysteines, but do not materially impairtheir relationship in the folded structure of the dimeric morphogenprotein, including their ability to form such intra- or inter-chaindisulfide bonds as may be necessary for morphogenic activity.Functionally equivalent sequences further include those wherein one ormore amino acid residues differs from the corresponding residue of areference sequence, e.g., the C-terminal seven cysteine domain (alsoreferred to herein as the conserved seven cysteine skeleton) of humanOP-1, provided that this difference does not destroy bone morphogenicactivity. Accordingly, conservative substitutions of corresponding aminoacids in the reference sequence are preferred. Amino acid residues thatare conservative substitutions for corresponding residues in a referencesequence are those that are physically or functionally similar to thecorresponding reference residues, e.g., that have similar size, shape,electric charge, chemical properties including the ability to formcovalent or hydrogen bonds, or the like. Particularly preferredconservative substitutions are those fulfilling the criteria defined foran accepted point mutation in Dayhoff et al. (1978), 5 Atlas of ProteinSequence and Structure, Suppl. 3, ch. 22 (pp. 354-352), Natl. Biomed.Res. Found., Washington, D.C. 20007, the teachings of which areincorporated by reference herein.

Natural-sourced osteogenic protein in its mature, native form is aglycosylated dimer typically having an apparent molecular weight ofabout 30-36 kDa as determined by SDS-PAGE. When reduced, the 30 kDaprotein gives rise to two glycosylated peptide subunits having apparentmolecular weights of about 16 kDa and 18 kDa. In the reduced state, theprotein has no detectable osteogenic activity. The unglycosylatedprotein, which also has osteogenic activity, has an apparent molecularweight of about 27 kDa. When reduced, the 27 kDa protein gives rise totwo unglycosylated polypeptides having molecular weights of about 14 kDato 16 kDa capable of inducing endochondral bone formation in a mammal.As described above, particularly useful sequences include thosecomprising the C-terminal 102 amino acid sequences of DPP (fromDrosophila), Vgl (from Xenopus), Vgr-1 (from mouse), the OP1 and OP2proteins, proteins (see U.S. Pat. No. 5,011,691 and Oppermann et al., aswell as the proteins referred to as BMP2, BMP3, BMP4 (see WO88/00205, U.S. Patent No. 5,013,649 and WO91/18098), BMP5 and BMP6 (see WO90/11366,PCT/US90/01630) and BMP8 and 9.

In certain preferred embodiments, bone morphogenic proteins usefulherein include those in which the amino acid sequences comprise asequence sharing at least 70% amino acid sequence homology or“similarity”, and preferably 80% homology or similarity with a referencemorphogenic protein selected from the foregoing naturally occurringproteins. Preferably, the reference protein is human OP-1, and thereference sequence thereof is the C-terminal seven cysteine domainpresent in osteogenically active forms of human OP-1, residues 330-431of SEQ ID NO: 2. Bone morphogenic proteins useful herein accordinglyinclude allelic, phylogenetic counterpart and other variants of thepreferred reference sequence, whether naturally-occurring orbiosynthetically produced (e.g., including “muteins” or “mutantproteins”), as well as novel members of the general morphogenic familyof proteins including those set forth and identified above. Certainparticularly preferred morphogenic polypeptides share at least 60% aminoacid identity with the preferred reference sequence of human OP-1, stillmore preferably at least 65% amino acid identity therewith.

In other preferred embodiments, the family of bone morphogenicpolypeptides useful in the present invention, and members thereof, aredefined by a generic amino acid sequence. For example, Generic Sequence7 (SEQ ID NO: 4) and Generic Sequence 8 (SEQ ID NO: 5) disclosed below,accommodate the homologies shared among preferred protein family membersidentified to date, including at least OP-1, OP-2, OP-3, CBMP-2A,CBMP-2B, BMP-3, 60A, DPP, Vgl, BMP-5, BMP-6, Vgr-l, and GDF-1. The aminoacid sequences for these proteins are described herein and/or in theart, as summarized above. The generic sequences include both the aminoacid identity shared by these sequences in the C-terminal domain,defined by the six and seven cysteine skeletons (Generic Sequences 7 and8, respectively), as well as alternative residues for the variablepositions within the sequence. The generic sequences provide anappropriate cysteine skeleton where inter- or intramolecular disulfidebonds can form, and contain certain critical amino acids likely toinfluence the tertiary structure of the folded proteins. In addition,the generic sequences allow for an additional cysteine at position 41(Generic Sequence 7) or position 46 (Generic Sequence 4), therebyencompassing the morphogenically active sequences of OP-2 and OP-3.

Generic Sequence 7 (SEQ ID NO:4)             Leu Xaa Xaa Xaa Phe Xaa Xaa              1               5 Xaa Gly Trp Xaa Xaa Xaa Xaa Xaa Xaa Pro         10                  15 Xaa Xaa Xaa Xaa Ala Xaa Tyr Cys Xaa Gly         20                  25 Xaa Cys Xaa Xaa Pro Xaa Xaa Xaa Xaa Xaa         30                  35 Xaa Xaa Xaa Asn His Ala Xaa Xaa Xaa Xaa         40                  45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa         50                  55 Xaa Xaa Xaa Cys Cys Xaa Pro Xaa Xaa Xaa         60                  65 Xaa Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa Xaa         70                  75 Xaa Xaa Xaa Val Xaa Leu Xaa Xaa Xaa Xaa         80                  85 Xaa Met Xaa Val Xaa Xaa Cys Xaa Cys Xaa         90                  95

wherein each Xaa independently is selected from a group of one or morespecified amino acids define as follows: “Res.” means “residue” and Xaaat res.2=(Tyr or Lys); Xaa at res.3=Val or Ile); Xaa at res.4=(Ser, Aspor Glu); Xaa at res.6=(Arg, Gln, Ser, Lys or Ala); Xaa at res.7=(Asp orGlu); Xaa at res.8=(Leu, Val or Ile); Xaa at res.11=(Gln, Leu, Asp, His,Asn or Ser); Xaa at res. 12=(Asp, Arg, Asn or Glu); Xaa at res.13=(Trpor Ser); Xaa at res.14=(Ile or Val); Xaa at res.15=(Ile or Val); Xaa atres.16 (Ala or Ser); Xaa at res.18=(Glu, Gln, Leu, Lys, Pro or Arg); Xaaat res.19=(Gly or Ser); Xaa at res.20=(Tyr or Phe); Xaa at res.21=(Ala,Ser, Asp, Met, His, Gln, Leu or Gly); Xaa at res.23=(Tyr Asn or Phe);Xaa at res.26=(Glu, His, Tyr, Asp, Gln, Ala or Ser); Xaa at res.28=(Glu,Lys, Asp, Gln or Ala); Xaa at res.30=(Ala, Ser, Pro, Gln, Ile or Asn);Xaa at res.31=(Phe, Leu, or Tyr); Xaa at res.33=(Leu, Val or Met); Xaaat res.34=(Asn, Asp, Ala, Thr or Pro); Xaa at res.35=(Ser, Asp, Glu,Leu, Ala or Lys); Xaa at res.36=(Tyr, Cys, His, Ser or Ile); Xaa atres.37=(Met, Phe, Gly or Leu); Xaa at res.38=(Asn, Ser, or Lys); Xaa atres.39=(Ala, Ser, Gly or Pro); Xaa at res.40=(Thr, Leu or Ser); Xaa atres.44=(Ile, Val or Thr); Xaa at res.45=(Val, Leu, Met or Ile); Xaa atres.46=(Gln or Arg); Xaa at res.47=(Thr, Ala or Ser); Xaa at res.48=(Leuor Ile); Xaa at res.49=(Val or Met); Xaa at res.50=(His, Asn or Arg);Xaa at res.51=(Phe, Leu, Asn, Ser, Ala or Val); Xaa at res.52=(Ile, Met,Asn, Ala, Val, Gly or Leu); Xaa at res.53=(Asn, Lys, Ala, Glu, Gly orPhe); Xaa at res.54=(Pro, Ser or Val); Xaa at res.55=(Glu, Asp, Asn,Gly, Val, Pro or Lys); Xaa at res.56=(Thr, Ala, Val, Lys, Asp, Tyr, Ser,Gly, Ile or His); Xaa at res.57=(Val, Ala or Ile); Xaa at res.58=(Pro orAsp); Xaa at res.59=(Lys, Leu or Glu); Xaa at res.60=(Pro, Val or Ala);Xaa at res.63=(Ala or Val); Xaa at res.65=(Thr, Ala or Glu); Xaa atres.66=(Gln, Lys, Arg or Glu); Xaa at res.67=(Leu, Met or Val); Xaa atres.68=(Asn, Ser, Asp or Gly); Xaa at res.69=(Ala, Pro or Ser); Xaa atres.70=(Ile, Thr, Val or Leu); Xaa at res.71=(Ser, Ala or Pro); Xaa atres.72=(Val, Leu, Met or Ile); Xaa at res.74=(Tyr or Phe); Xaa atres.75=(Phe, Tyr, Leu or His); Xaa at res.76=(Asp, Asn or Leu); Xaa atres.77=(Asp, Glu, Asn, Arg or Ser); Xaa at res.78=(Ser, Gln, Asn, Tyr orAsp); Xaa at res.79=(Ser, Asn, Asp, Glu or Lys); Xaa at res.80=(Asn, Thror Lys); Xaa at res.82=(Ile, Val or Asn); Xaa at res.84=(Lys or Arg);Xaa at res.85=(Lys, Asn, Gln, His, Arg or Val); Xaa at res.86=(Tyr, Gluor His); Xaa at res.87=(Arg, Gln, Glu or Pro); Xaa at res.88=(Asn, Glu,Trp or Asp); Xaa at res.90=(Val, Thr, Ala or Ile); Xaa at res.92=(Arg,Lys, Val, Asp, Gln or Glu); Xaa at res.93=(Ala, Gly, Glu or Ser); Xaa atres.95=(Gly or Ala) and Xaa at res.97=(His or Arg).

Generic Sequence 8 (SEQ ID NO: 5) includes all of Generic Sequence 7(SEQ ID NO: 4) and in addition includes the following sequence (SEQ IDNO: 6) at its N-tenninus:

Cys Xaa Xaa Xaa Xaa   1              10 5

Accordingly, beginning with residue 7, each “Xaa” in Generic Sequence 8is a specified amino acid defined as for Generic Sequence 7 (SEQ ID NO:4), with the distinction that each residue number described for GenericSequence 7 (SEQ ID NO: 4) is shifted by five in Generic Sequence 8 (SEQID NO: 5). Thus, “Xaa at res.2=(Tyr or Lys)” in Generic Sequence 7refers to Xaa at res. 7 in Generic Sequence 8 (SEQ ID NO: 5). In GenericSequence 8 (SEQ ID NO: 5), Xaa at res.2=(Lys, Arg, Ala or Gin); Xaa atres.3=(Lys, Arg or Met); Xaa at res.4=(His, Arg or Gin); and Xaa atres.5=(Glu, Ser, His, Gly, Arg, Pro, Thr, or Tyr).

In another embodiment, useful osteogenic proteins include those definedby Generic Sequences 9 (SEQ ID NO: 8) and 10 (SEQ ID NO: 7), describedherein above.

As noted above, certain currently preferred bone morphogenic polypeptidesequences useful in this invention have greater than 60% identity,preferably greater than 65% identity, with the amino acid sequencedefining the preferred reference sequence of hOP-1. These particularlypreferred sequences include allelic and phylogenetic counterpartvariants of the OP-1 and OP-2 proteins, including the Drosophila 60Aprotein. Accordingly, in certain particularly preferred embodiments,useful morphogenic proteins include active proteins comprising pairs ofpolypeptide chains within the generic amino acid sequence hereinreferred to as “OPX” (SEQ ID NO: 3), which defmes the seven cysteineskeleton and accommodates the homologies between several identifiedvariants of OP-1 and OP-2. As described therein, each Xaa at a givenposition independently is selected from the residues occurring at thecorresponding position in the C-terminal sequence of mouse or human OP-1or OP-2.

In still another preferred embodiment, useful osteogenically activeproteins have polypeptide chains with amino acid sequences comprising asequence encoded by nucleic acid that hybridizes, under low, medium orhigh stringency hybridization conditions, to DNA or RNA encodingreference morphogen sequences, e.g., C-terminal sequences defining theconserved seven cysteine domains of OP-1, OP-2, BMP2, 4, 5, 6, 60A,GDF3, GDF6, GDF7 and the like. As used herein, high stringenthybridization conditions are defined as hybridization according to knowntechniques in 40% formamide, 5×SSPE, 5×Denhardt's Solution, and 0.1% SDSat 37° C. overnight, and washing in 0.1×SSPE, 0.1% SDS at 50° C.Standard stringence conditions are well characterized in commerciallyavailable, standard molecular cloning texts.

As noted above, proteins useful in the present invention generally aredimeric proteins comprising a folded pair of the above polypeptides.Such morphogenic proteins are inactive when reduced, but are active asoxidized homodimers and when oxidized in combination with others of thisinvention to produce heterodimers. Thus, members of a folded pair ofmorphogenic polypeptides in a morphogenically active protein can beselected independently from any of the specific polypeptides mentionedabove.

The bone morphogenic proteins useful in the materials and methods ofthis invention include proteins comprising any of the polypeptide chainsdescribed above, whether isolated from naturally-occurring sources, orproduced by recombinant DNA or other synthetic techniques, and includesallelic and phylogenetic counterpart variants of these proteins, as wellas biosynthetic variants (muteins) thereof, and various truncated andfusion constructs. Deletion or addition mutants also are envisioned tobe active, including those which may alter the conserved C-terminal sixor seven cysteine domain, provided that the alteration does notfunctionally disrupt the relationship of these cysteines in the foldedstructure. Accordingly, such active forms are considered the equivalentof the specifically described constructs disclosed herein. The proteinsmay include forms having varying glycosylation patterns, varyingN-termini, a family of related proteins having regions of amino acidsequence homology, and active truncated or mutated forms of native orbiosynthetic proteins, produced by expression of recombinant DNA in hostcells.

The bone morphogenic proteins contemplated herein can be expressed fromintact or truncated cDNA or from synthetic DNAs in prokaryotic oreukaryotic host cells, and purified, cleaved, refolded, and dimerized toform morphogenically active compositions. Currently preferred host cellsinclude E. coli or mammalian cells, such as CHO, COS or BSC cells.Detailed descriptions of the bone morphogenic proteins useful in thepractice of this invention, including how to make, use and test them forosteogenic activity, are disclosed in numerous publications, includingU.S. Pat. Nos. 5,266,683 and 5,011,691, the disclosures of which areincorporated by reference herein.

Thus, in view of this disclosure, skilled genetic engineers can isolategenes from cDNA or genomic libraries of various different biologicalspecies, which encode appropriate amino acid sequences, or constructDNAs from oligonucleotides, and then can express them in various typesof host cells, including both prokaryotes and eukaryotes, to producelarge quantities of active proteins capable of stimulating endochondialbone morphogenesis in a mammal.

B. Preparations of Bone Morphogenic Protein, OP-1 1. Lyophilized Protein

OP-1 can be lyophilized from 20 mM acetate buffer, pH 4.5, with 5%mannitol, lactose, glycine or other additive or bulking agent, usingstandard lyophilization protocols. OP-1 reconstituted in this manner hasbeen observed to be biologically active for at least six months storedat 4° C. or 30° C.

OP-1 can also be lyophilized from a succinate or a citrate buffer (orother non-volatile buffer) for re-constitution in water, and from waterfor re-constitution in 20 mM acetate buffer, pH 4.5. Generally,additives such as lactose, sucrose, glycine and mannitol are suitablefor use in lyophilized matrix-free osteogenic devices. In certainembodiments, such devices (0.5 mg/ml OP-1 and 5% additive) can beprepared in a wet or dry configuration prior to lyophilization.

For example, liquid formulations of OP-1 in 10 and 20 mM acetate buffer(pH 4, 4.5 and 5) with and without mannitol (0%, 1% and 5%) are stableand osteogenically active for at least six months.

II. CARRIER CONSIDERATIONS

As already explained, “carrier” as used herein means a biocompatible,non-rigid, amorphous material having no defined surfaces suitable foruse with the devices, implants and methods of the present invention.Suitable carriers are non-particulate and are non-porous, i.e., arepore-less. Carriers suitable for use in the instant invention lack ascaffolding structure and are substantially matrix-free. Thus,“substantially free of matrix” is also understood to mean that, when acarrier-containing device is provided to a defect locus, no substratecompetent to act as a scaffold per se is introduced from an exogenoussource, including the carrier. Prior to delivery to and implantation inthe recipient, the carrier is recognized by virtue of its chemicalnature to be substantially unable to contribute a three-dimensionalscaffolding structure to the device. Preferred carriers are adherent, atleast transiently, to tissues such as bone, cartilage and/or muscles.Certain preferred carriers are water-soluble, viscous, and/or inert.Additionally, preferred carriers do not contribute significant volume toa device. Currently preferred carriers are selected from the groupconsisting of: alkylcelluloses, poloxamer, gelatins, polyethyleneglycols (PEG), dextrins, vegetable oils and sugars. Particularlypreferred carriers currently include, but are not limited to, PluronicF127™ poloxamer, carboxymethylcelluloses (CMC), (e.g., low viscosity CMCfrom Aqualon), lactose, PEG, mannitol, sesame oil, and hetastarch(Hespan, Dupont), and combinations thereof. Other preferred carriersinclude, without limitation, acetate buffer (20 mM, pH 4.5),physiological saline, and citrate buffers. In the case of devicescomprising carriers such as acetate, poloxamers and PBS, administrationby injection can result in precipitation of certain osteogenic proteinsat the administration site.

III. FORMULATION AND DELIVERY CONSIDERATIONS

The devices of the invention can be formulated using routine methods.All that is required is determining the desired final concentration ofosteogenic protein per unit volume of carrier, keeping in mind that thedelivered volume of device will be less than the volume the void at thedefect locus. The desired final concentration of protein will depend onthe specific activity of the protein as well as the type, volume, and/oranatomical location of the defect. Additionally, the desired finalconcentration of protein can depend on the age, sex and/or overallhealth of the recipient. Typically, for a critical-sized segmentaldefect approximately at least 2.5 cm in length, 0.05 ml (or mg) of adevice containing 0.5-1.5 mg osteogenic protein has been observed toinduce bone formation sufficient to repair the gap. In the case of anon-critical sized defect fresh fractures, approximately 0.1-0.5 mgprotein has been observed to repair the gap or defect. Optimization ofdosages requires no more than routine experimentation and is within theskill level of one of ordinary skill in the art.

As exemplified below, the devices of the present invention can assume avariety of configurations. For example, a matrix-free osteogenic devicein solution can be formulated by solubilizing certain forms of OP-1 insolutions of acetate (20 mM, pH4.5) or citrate buffers, orphosphate-buffered saline (PBS), pH 7.5. In some instances, theosteogenic protein may not be entirely solubilized and/or mayprecipitate upon administration into the defect locus. Suspensions,aggregate formation and/or in vivo precipitation does not impair theoperativeness of the matrix-free osteogenic device when practiced inaccordance with the invention disclosed herein. Matrix-free devices insolution are particularly suitable for administration by injection, suchas providing a device to a fracture locus by injection rather thansurgical means.

Generally speaking, the configuration of matrix-free devices suitablefor delivery by injection differ from those preferred for use at anopen, surgical site. For example, lyophilized preparations ofmatrix-free devices are one currently preferred embodiment for repair ofthis type of defect. The above-described matrix-free devices in solutioncan be used to prepare a lyophilized configuration. For example, asdescribed below, the osteogenic protein OP-1 can be admixed with theabove-described buffers and then lyophilized. OP-1 can also belyophilized from mannitol-containing water.

As exemplified below, lyophilized configurations of matrix-freeosteogenic devices can induce bone formation in critical-sized andnon-critical sized segmental defects. For example, providing alyophilized device to a segmental defect locus comprises depositingnon-contiguous aliquots of the lyophilized device along the length ofexposed muscle spanning the segmental defect, such that the total numberof aliquots provides an amount of osteogenic protein sufficient toinduce bone formation which ultimately fills the void at the defectlocus. Placement is followed by routine closure of the defect sitewhereby the layers of muscles and associated tissue are sutured,layer-by-layer, to enclose the aliquots in the void at the defect locus.This type of delivery and surgical closure require only routine skilland experimentation. Similar formulations and methods of delivery can beused to induce bone formation for repair of a gap caused by a failedprosthetic, bone tumor resection, cranial/facial reconstruction, spinalfusions and massive allograft defects. Any modifications of theabove-described methods of delivery which may be required forspecialized applications of lyophilized matrix-free devices are withinthe skill level of the artisan and require only routine experimentation.

Yet another configuration of matrix-free devices is exemplified below.Osteogenic protein and a carrier such as carboxymethylcellulose (lowviscosity, Aqualon, or Pluronic F127™ poloxamer can be admixed to form apaste. In some embodiments, approximately saline is added to carrier toform a paste into which an osteogenic protein such as OP-1 can bedispersed. A paste configuration can be used to paint the surfaces of adefect such as a cavity. Pastes can be used to paint fracture defects,chondral or osteochondral defects, as well as bone defects at aprosthetic implant site. A paste can also be injected or extruded intoor along one of the surfaces of a defect, in a manner similar toextruding toothpaste or caulking from a tube, such that a bead ofmatrix-free device is delivered along the length of the defect locus.Typically, the diameter of the extruded bead is determined by the typeof defect as well as the volume of the void at the defect locus.

Carriers such as carboxymethylcellulose can also be used to formulate adevice with a configuration like putty. As will be obvious to theskilled artisan, such a configuration results from adjusting theproportion of carrier to wetting agent, with less wetting agentproducing a drier device and more producing a wetter device. The precisedevice configuration suitable to repair a defect will at least depend onthe type of defect and the size of the defect. The skilled artisan willappreciate the variables.

Yet another configuration which is suitable for the devices of theinstant invention is a gel. This is exemplified below by thepoloxamer-containing matrix-free devices which can induce bone formationin vivo. Gels of this type have been used to treat fractures as well asgap repairs. One useful feature of this configuration is that theviscosity of the gel can be manipulated by adjusting the amount ofcarrier, thereby permitting a wide-range of applications (e.g.,segmental defects, fractures, and reconstructions) and modes of delivery(e.g., injection, painting, extrusion, and the like). Thus this type ofdevice can assume at least the forms of an injectable liquid, a viscousliquid, and an extrudable gel merely by manipulating the amount ofcarrier into which the osteogenic protein is dispersed.

In yet other embodiments of the present invention, preparation of theactual osteogenic device can occur immediately prior to its delivery tothe defect locus. For example, CMC-containing devices can be preparedon-site, suitable for admixing immediately prior to surgery. In oneembodiment, low viscosity CMC (Aqualon) is packaged and irradiatedseparately from the osteogenic protein OP-1. The OP-1 protein then isadmixed with the CMC carrier, and tested for osteogenic activity.Devices prepared in this manner were observed to be as biologicallyactive as the conventional device without CMC. Again, all that isrequired is determining the effective amount of osteogenic protein toinduce bone formation sufficient to fill the defect locus andmaintaining a device volume which is less than the volume of the void atthe defect locus. The precise manner in which the device of the presentinvention is formulated, and when or how formulation is accomplished, isnot critical to operativeness.

Practice of the invention will be still more fully understood from thefollowing examples, which are presented herein for illustration only andshould not be construed as limiting the invention in any way.

IV. BIOASSAY A. Bioassay of Osteogenic Activity: Endochondral BoneFormation and Related Properties

The following sets forth protocols for identifying and characterizingbona fide osteogenic or bone morphogenic proteins as well as osteogenicdevices within the scope of Applicants' invention.

The art-recognized bioassay for bone induction as described by Sampathand Reddi (Proc. Natl. Acad. Sci. USA (1983) 80:6591-6595) and U.S. Pat.No. 4,968,590, the disclosures of which are herein incorporated byreference, is used to establish the efficacy of the purificationprotocols. As is demonstrated below, this assay consists of depositingthe test samples in subcutaneous sites in allogeneic recipient ratsunder ether anesthesia. A vertical incision (1 cm) is made under sterileconditions in the skin over the thoracic region, and a pocket isprepared by blunt dissection. In certain circumstances, approximately 25mg of the test sample is implanted deep into the pocket and the incisionis closed with a metallic skin clip. The heterotropic site allows forthe study of bone induction without the possible ambiguities resultingfrom the use of orthotopic sites.

The sequential cellular reactions occurring at the heterotropic site arecomplex. The multistep cascade of endochondral bone formation includes:binding of fibrin and fbronectin to implanted matrix, chemotaxis ofcells, proliferation of fibroblasts, differentiation into chondroblasts,cartilage formation, vascular invasion, bone formation, remodeling, andbone marrow differentiation.

In rats, this bioassay model exhibits a controlled progression throughthe stages of matrix induced endochondral bone development including:(1) transient infiltration by polymorphonuclear leukocytes on day one;(2) mesenchymal cell migration and proliferation on days two and three;(3) chondrocyte appearance on days five and six; (4) cartilage matrixformation on day seven; (5) cartiliage calcification on day eight; (9)vascular invasion, appearance of osteoblasts, and formation of new boneon days nine and ten; (10) appearance of osteoblastic and boneremodeling on days twelve to eighteen; and (11) hematopoietic bonemarrow differentiation in the ossicle on day twenty-one.

Histological sectioning and staining is preferred to determine theextent of osteogenesis in the implants. Staining with toluidine blue orhemotoxylin/eosin demonstrates clearly the ultimate development ofendochondrial bone. Twelve day bioassays are usually sufficient todetermine whether bone inducing activity is associated with the testsample.

Additionally, alkaline phosphatase activity can be used as a marker forosteogenesis. The enzyme activity can be determinedspectrophotometrically after homogentization of the excised testmaterial. The activity peaks at 9-10 days in vivo and thereafter slowlydeclines. Samples showing no bone development by histology should haveno alkaline phosphatase activity under these assay conditions. The assayis useful for quantitation and obtaining an estimate of bone formationvery quickly after the test samples are removed from the rat. Forexample, samples containing osteogenic protein at several levels ofpurity have been tested to determine the most effective dose/puritylevel, in order to seek a formulation which could be produced on anindustrial scale. The results as measured by alkaline phosphataseactivity level and histological evaluation can be represented as “boneforming units”. One bone forming unit represents the amount of proteinthat is need for half maximal bone forming activity on day 12.Additionally, dose curves can be constructed for bone inducing activityin vivo at each step of a purification scheme by assaying variousconcentrations of protein. Accordingly, the skilled artisan canconstruct representative dose curves using only routine experimentation.

B. Bone Formation Following Implantation of Matrix-Free OP-1 OsteogenicDevices

Osteogenic devices were made with 62.5 μg lyophilized OP-1, either withor without 25 mg collagen matrix. These devices were evaluated for theirability to support bone formation using the above-described rat ectopicbone formation assay, in both an intramuscular and subcutaneous site.Additionally, the mass of bone formed was assessed by measuring thecalcium contents and the weights of the removed devices. The datagenerated is summarized in Tables 1 and 2 below.

As evidenced by both histology and calcium content, bone formed inresponse to all of the matrix-free OP-1 samples. These data illustratethat implanting a matrix-free OP-1 device alone is sufficient to induceendochondral bone formation in rat ectopic sites.

TABLE 1 Bone Formation vs. Concentrations of OP-1 Intramuscular SiteDevice 12 Day Implant ( ) Sample Size Calcium Implant Histology,Collagen OP-1 μg/mg tissue Weight, mg % bone 25 mg (5) 62.5 μg 38.2-67.170-90  0 mg 51.7 μg 48.6 430.7 mg 90  0 mg 61.3 μg 35.8 256.1 mg 90  0mg 18.6 μg 66.3 457.8 mg 90  0 mg 59.2 μg 53.9 580.0 mg 90  0 mg 59.5 μg25.9 383.1 mg 90

TABLE 2 Bone Formation vs. Concentrations of OP-1 Subcutaneous SiteDevice 12 Day Implant ( ) Sample Size Calcium Implant Histology,Collagen OP-1 μg/mg tissue Weight, mg % bone 25 mg (5) 62.5 μg 28.4-54.6 500.6-1472.6 60-80  0 mg 40.4 μg 44.4 132.7 mg 90  0 mg 36.7 μg 43.9337.7 mg 80  0 mg 35.2 μg 37.4 310.7 mg 90  0 mg 51.0 μg 57.5 195.8 mg90  0 mg 51.4 μg 29.7 721.9 mg 90

C. Matrix-Free OP-1 Devices Containing Water-Soluble Carriers

OP-1 admixed with water soluble carriers also support bone formation. Inthis study, mannitol, carboxymethylcellulose, dextrin, PEG3350, aPluronic gel and collagen each were formulated into a paste by theaddition of 0.9% sterile saline. Ten μg of OP-1 dissolved in water wasadded to the paste to produce a matrix-free device, and the device wasimmediately implanted intramuscularly in rats. After 12 days, theimplanted devices were removed and evaluated for bone formation by bothcalcium content and histology. These data confirm the above-describedobservation that collagen matrix is not essential for inducingvolume-filling bone formation. Of the water-soluble carriers evaluated,mannitol demonstrated the best results overall, with the othersappearing relatively comparable.

TABLE 3 Histology Calcium, μg/mg implant Implant ug, mg (%) Mannitol100-200 28-48 >90 CMC  50-200 28-52 >80-90 Dextrin 20-80  8-12 >90Collagen 550-800 20-45 >90 PEG 100-150 10-12 >90 Poloxamer  50-150 14-28>90

D. Matrix-Free OP-1 Devices in Solution

Matrix-free osteogenic devices in solution also were demonstrated toinduce bone formation when administered either intramuscularly (IM), orintradermally (ID). In an exemplary experiment, matrix-free OP-1 deviceswere prepared in 20 mM acetate buffer, pH 4.5. The devices were preparedsuch that the desired dose of (5-50 mg) OP-1 would be delivered in a 100μL injection volume. Both forms of administration induced bone formationas measured by calcium content, histology and explant weight.

E. Poloxamer Gel-Containing Matrix-Free OP-1 Devices

Experiments currently preferred were initiated with a poloxamerformulation of the matrix-free OP-1 device. In one embodiment, thisdevice has the unique property of being liquid at refrigerationtemperatures, but gel-like when warmed to room temperature. This allowsthe device to be drawn up into syringe when cold, allowing for easyinjection after a few minutes at room temperature. This was useful forinjecting such OP-1 devices into fracture sites, since the gel permitscontainment of OP-1 at the site of injury.

Poloxamer gel, prepared from 0.5 g of commercially available PluronicF127™ poloxamer to which 1.35 mL of water is added, is a viscous liquidat refrigeration temperature and a semisolid gel at room temperature.Bone formation was observed after 12 days in response to IMadministration of matrix-free OP-1-Pluronic gel devices. The gel was notinjected directly into the muscle, but was injected into the muscle flapwithout the use of a needle. Bone also formed in response to SCadministration of these same poloxamer gel devices. The OP-1 dose (5-25mg) was contained in 50 μl gel.

Recovery of OP-1 from the gel devices was determined by extracting with8M urea buffer and injecting the extract onto the HPLC. 100% recovery ofthe OP-1 from the gel was obtained. In addition, urea extracts ofselected gels were as active as an OP-1 standard. For example, one ofthe gels was re-extracted after 10 days storage at refrigerationtemperature in a syringe; 100% recovery of OP-1 was obtained and againthe extract was as active as an OP-1 standard.

As described below, sterile filtered OP-1 can be added to autoclaved orirradiated gel in an aspectic manner so that a sterile device can beprovided.

OP-1 has been admixed with autoclaved poloxamer gels. The gel wasprepared, autoclaved and then chilled so that it liquified prior toadmixture with the OP-1. The OP-1 gel was then filled into syringeswhich were stored at 5° C. The samples were observed to be stable for atleast 2 weeks at 5° C. Approximately at least 50-60% of the initial OP-1remained after 7 weeks storage at 5° C. Gels were also prepared fromirradiated Pluronic F127™ poloxamer. Recoveries of 40-50% was observedafter 7 weeks at 5° C.

A second time course was carried out for the purposes of evaluating boneformation in response to matrix-free OP-1-poloxamer gel devices. 50 μlvolumes of poloxamer gel containing zero or 10 μg of OP-1 were injectedinto a muscle flap. Implants removed at day 7, 12 and 21 were analyzedfor calcium content and alkaline phosphatase activity. The time courseof bone formation was similar to that observed with the standardcollagen-containing OP-1 device.

V. ANIMAL STUDIES: METHODS OF USE OF MATRIX-FREE OP-1 DEVICES ANDIMPLANTS A. Healing of Critical-Sized Segmental Defects in Dogs UsingMatrix-Free Osteogenic Devices 1. Experiment 1

The following experiments demonstrate the efficacy of injectable andfreeze-dried formulations of rhOP-1 for healing both critical andnon-critical sized segmental defects in an established canine ulnadefect model. Three formulations of matrix-free osteogenic devices, eachcontaining OP-1, were evaluated in critical-size defects (2.5 cm) and/ornon-critical size defects (5 mm, 3 mm, 1.5 mm). As described above,critical size defects are defects which will not heal spontaneously. Thethree formulations evaluated were (1) 20 mm acetate buffered solution(pH 4.5); (2) phosphate-buffered saline (PBS, approximately pH 7.5); and(3) lyophilized (freeze-dried) protein alone. The amount of OP-1provided in the critical size defects was 1.75 mg; 0.35 mg protein wasprovided in the non-critical size defects. Immediately before woundclosure, OP-1 was admixed with acetate or PBS and then injected at thedefect site in a total of 1 ml. Lyophilized samples were placed alongthe length of the defect in 5 separate aliquots at discrete,non-contiguous loci along the length of the defect.

Using standard surgical techniques, osteoperiosteal segmental defects ofthe prescribed size were created bilaterally in the mid ulna region oftwenty adult bred-for-purpose mongrel dogs. All animals were between oneand two years old, weighed from 35 to 50 pounds, and were supplied byUSDA licensed providers. Special attention was paid in selecting animalsof uniform size and weight to limit the variability in bone geometry andloading. The animals were radiographically screened preoperatively toensure proper size, skeletal maturity, and that no obvious osseousabnormalities existed. The animals also were screened clinically toexclude acute and chronic medical conditions during a two-weekquarantine period. A complete blood count with cell differential wasperformed prior to surgery.

The radius was maintained for mechanical stability, but no internal orexternal fixation was used. The site was irrigated with saline to removebone debris and spilled marrow cells and then dried and homeostasis wasachieved prior to providing the formulation to the site. Thesoft-tissues were meticulously closed in layers before injection offormulations 1 or 2 (acetate or PBS). Formulation 3 devices (lyophilizedprotein alone) was placed along the interosseous space before closingthe tissue layers to contain the implant. The procedure was thenrepeated on the contralateral side, except that no OP-1 device wasprovided before wound closure.

Animals were administered intramuscular antibiotics for four dayspost-surgery and routine anterior-posterior radiographs were takenimmediately after surgery to insure proper placement. Animals were keptin 3×4 recovery cases for 24 to 72 hours postoperatively after whichthey were transferred to runs and allowed unrestricted motion.

Biweekly radiographs were taken to study the progression of healing. Inaddition, pre-operative blood (serum) was taken biweekly until sacrificeto study antibody formation by the sponsor. At sacrifice, all ulnae wereretrieved en bloc and those that were healed sufficiently weremechanically tested in torsion. Segments were evaluated by histology fortissue response, bone architecture and remodeling, and quality andamount of new bone formation and healing.

Animals were sacrificed at 4, 6, 8 or 12 weeks post operatively.

1b(3). Radiographs

Radiographs of the forelimbs were obtained biweekly until eight weekspostoperative and then again at sacrifice at twelve postoperative weeks.Standardized exposure times and intensities were used, and sandbags wereused to position the extremities in a consistent manner. Radiographswere evaluated and compared to earlier radiographs to appreciate qualityand speed of defect healing.

Mechanical Testing

Immediately after sectioning, if healing was deemed sufficient by manualmanipulation, specimens were tested to failure in torsion on an MTSclosed-loop hydraulic test machine (Minneapolis, Minn.) operated instroke control at a constant displacement rate of 50 mm/min in acylindrical aluminum sleeve and cemented with methylmethacrylate usingmanufacturer's protocol. One end was rigidly fixed and the other wasrotated counterclockwise. Since the dog ulna has a slight curvature, thespecimens were mounted eccentrically to keep specimen rotation coaxialwith that of the testing device. The torsional force was applied with alever arm of six cm, by a servohydraulic materials testing system.Simultaneous recordings were made of implant displacement, as measuredby the machine stroke controller, while load was recorded from the loadcell. Data was recorded via an analog-to-digital conversion board and apersonal computer and an online computer acquisition software.Force-angular displacement curves were generated from which the torqueand angular deformation to failure were obtained, and the energyabsorption to failure computed as the area under the load-displacementcurve.

Results

The bone healing characteristics, mechanical strength, and histology ofcritical size ulna defects treated with rhOP-1 without carrier materialwere similar to that of defects treated with the standard OP-1 device.In brief, the experimental observations were as follows: New boneformation and healing patterns observed radiographically in defectstreated with rhOP-1 without a matrix were similar to healing patternsobserved previously with the conventional collagen-containing OP-1device. In general, new bone formation was evident as early as two weekspostoperative. The new bone continued to densify, consolidate andremodel until sacrifice at twelve postoperative weeks. This studydemonstrated that functional bony union is possible with human OP-1devices without matrix. Additionally, the gross appearance and thetwelve week histologic characteristics were similar to that observedwith the conventional collagen-containing OP-1 device. Of the sixdefects treated with a matrix-free OP-1 device, four had solid bonyunions at twelve weeks postoperative. The remaining two defects, in thesame animal, demonstrated some early new bone formationradiographically, however at sacrifice were incompletely spanned orfilled with new bone. The mean torsional load to failure of the healeddefects was 40.05N (This represents the equivalent of about 79% ofpreviously tested segmental defects treated with the traditionalcollagen-containing OP-1 device and 61% of previously tested intactcontrol ulna.)

Radiographically, extensive new bone formation was observed at two weekspostoperative with all three formulations. From two to 12 weeks, newbone increased in volume and radiodensity and filled and spanned thedefects. By 12 weeks postoperative (sacrifice), radiodense new bone hadsignificantly filled and bridged the defects treated with rhOP-1formulations. All rhOP-1 defects were mechanically stable and bridgedwith new bone at sacrifice at 12 weeks. No significant differences werefound among the three formulations for radiographic appearance, althoughFormulations 1 and 2 scored slightly higher than Formulation 3 at alltime periods.

Histologically, proliferative new bone was present within andsurrounding the defects treated with rhOP-1. Bridging of the defects andbony healing was significantly completed by 12 weeks postoperative withgaps of fibrocartilage between areas of significant new bone growth.Signs of early cortex development with densification of the new boneborders was present in all rhOP-1 defects. There were no differences inhistologic grading results based upon formulation type, althoughFoundation 2 scored highly than Formulations 3 and 1 for quality ofunion.

The mean load to failure of the Formulation 1 defects was 47.64N (94% ofdefects treated with the standard OP-1 device and 73% of previouslytested intact controls). The mean load to failure of the Formulation 2defects was 51.96N (102% of defects treated with the standard OP-1device and 80% of previously tested intact controls). The mean load tofailure of the Formulation 3 defects was 50.86N (100% of defects treatedwith the standard OP-1 device and 78% of previously tested intactcontrols). No significant differences were noted in the mechanicaltesting results among formulation type. Formulation 2 defects scoredslightly higher than Formulations 3 and 1 in maximum load to failure.

TABLE 4 Mechanical testing results for critical and non-critical sizedefects treated with rhOP-1 versus nontreated controls. Means ± SD(Sample size). Defect Size Max Load Torque % of Intact (Treatment) (N)(Nm) Ulna 2.5 cm 46.11± 2.77± 70.64± (rhOP-1) 17.42(10) 1.05(10)26.69(10) 2.5 cm * * * (control) 5.0 mm 51.81± 3.11± 79.37± (rhOP-1)17.94(6) 1.08(6) 27.48(6) 5.0 mm  9.20± 0.55± 14.09± (control)  6.62(5)0.40(5) 10.14(5) 3.0 mm 64.10± 3.85± 98.21± (rhOP-1) 49.12(4) 2.95(4)75.25(4) 3.0 mm 21.91± 1.31± 33.57± (control) 26.46(3) 1.59(3) 40.54(3)1.5 mm 62.17± 3.73± 95.25± (rhOP-1) 30.48(4) 1.83(4) 46.70(4) 1.5 mm19.43± 1.17± 29.76± (control) 19.11(4) 1.15(4) 29.28(4) * Defects nottested due to instability.

Formulations using 20 mm acetate buffer, pH 4.5 and 5% mannitol alsowere tested. Studies using non-critical size defects (5 mm gaps) showeda clear increase in the rate of healing when OP-1 was present, ascompared with controls. Specifically, at 8 weeks, the controls averaged14% the strength of intact ulnas, while the OP-1 treated defectsaveraged 79%. Radiographically, new bone was evident as early as twoweeks postoperative and by eight weeks significantly began to fill andbridge the defects treated with rhOP-1 formulations. None of the sixnontreated control defects were completely healed at the end of thestudy period. One of three defects with Formulation 1 (acetate buffer)and three of three defects treated with Formulation 2 (PBS) werecompletely filled and bridged by new bone at 8 weeks. All six defectsreceiving OP-1 had new bone formation and were mechanically stable.Formulation 2 defects scored significantly higher in radiographicgrading results, energy absorbed to failure, and histologic quality ofunion that defects treated with Formulation 1. Otherwise, no differenceswere fund between the two formulations for mean load to failure, angulardeformation and overall histologic appearance. Both formulations scoredsignificantly higher in all categories compared to control defects.Histologically, proliferative new bone was present within andsurrounding the defects treated with rhOP-1. Bridging of the defects andbony healing was almost completed by eight weeks postoperative with gapsof fibrocartilage between areas of significant new bone growth. Evidenceof early cortex development and bone remodeling was present in some ofthe rhOP-1 defects. All nontreated controls resulted in incompleteunions although potential longer term healing was indicated by some newbone formation fro the host bone ends and endosteal regions. The meanload to failure of the Formulation 2 defects was 53.23N (104.5% ofcritical size defects treated with the standard OP-1 device and 81.6% ofpreviously tested intact controls). Comparison of non-critical sizedefects treated with 0.35 mg OP-1 to previously tested critical sizedefects treated with 1.5 mg OP-1 without a carrier material (InjectableFormulations) showed no significant difference in mean load to failure.

C. Healing of Fracture Defects Using Matrix-Free Osteogenic Devices 1.Rabbit Fracture Study

A rabbit fracture repair model study (ulna midshaft fracture) alsodemonstrates the efficacy of the methods and devices of the invention.This study compared the effect of administration of matrix-free OP-1devices in three configurations: 1) acetate buffer pH 4.5 (solubleOP-1), 2) PBS (suspension OP-1) and 3) poloxamer gel. Four rabbits weretreated in each group immediately after fracture creation; contralateralcontrols were no-defect arms. Animals were sacrificed 3 weeks posttreatment. In summary, animals injected with the acetate- or poloxamer-contanining OP-1-devices showed a significantly larger fracture callusby radiographic, gross and histological examination. The mean torsionalload to failure for all ulna treated with OP-1 was 8.89±2.99 N(mean±standard deviation) (8 samples). While the mean load to failurefor non-treated control ulna was 7.9±2.92 N (9 samples).

1a. Test Material Description

Matrix-free OP-1 devices in solution were utilized. The three solutionconfigurations evaluated were: (1) rhOP-1 admixed with phosphatebuffered saline, 8.71 mg OP- 1/ml. The devices were packaged inindividual vials. The estimated range of device volume delivered wasbetween 30 μl and 110 μl per site; (2) rhOP-1 admixed with 20 mM acetatebuffer, pH 4.5, 0.99 mg OP-1/ml. The devices were packaged in individualvials containing 130 μl. The device was drawn up into a syringe. In allcases less than 100 μl was delivered to each site. The estimated rangeof implant volume delivered was between 60 μl and 90 μl per site; and(3) rhOP-1 in Pluronic gel, 0.87 mg OP-1/ml. This device was packaged ina syringe. The device was kept refrigerated until administration to thedefect site (lapse time less than one minute). All Thick gel wasdelivered in all cases using a large gauge (18) needle.

In all cases, dosages were calibrated to deliver approximately 100 μg ofrhOP-1 to each fracture site.

A total of twelve adult male rabbits, adult male White New Zealandrabbits bred for purpose, at least 9 months of age at onset of studywere utilized. All animals were skeletally mature and weighed between2.4 and 3.0 kg were supplied by USDA licensed vendors. The animals werescreened to exclude acute and chronic medical conditions during aquarantine period, and were radiographically screened to ensure propersize, skeletal maturity, and that no obvious osseous abnormalitiesexist. Specific attention was paid to selecting animals of uniform sex,size and weight to limit the variability of healed fracture strength.Experimental traverse fractures were created bilaterally in the centerulna of each animal using standard surgical techniques. The left ulnaserved as an untreated control in each animal. Briefly, using standardaseptic techniques, bilateral fractures were induced by making lateralincision approximately 2.0 cm in length and exposing the right ulna wasobtained using sharp and blunt dissection. A transverse osteotomy wascreated in the mid-ulna using an electrical surgical saw. The site thenwas closed with resorbable suture. A matrix-free OP-1 device in solutionwas then injected through the soft tissues into the fracture site. Theprocedure was then repeated on the left side with the exception that noOP-1 device was provided to these fracture sites.

Animals were administered intramuscular antibiotics for four dayspost-surgery. Animals were kept in recovery cages postoperatively untilfully conscious and weight bearing, after which they were transferred tostandard cages and allowed unrestricted motion. The limbs were notcasted.

Weekly radiographs were taken to study the progression of healing. Allanimals were sacrificed at three postoperative weeks. All ulna wereretrieved en bloc and mechanically tested in torsion. Fracture healingwas further evaluated by histology for quality and amount of new boneformation and healing.

At one week postoperative, early new bone formation was evident in allfractures. Traces of lightly radiodense material was present along theperiosteal borders. The amount of new bone formation was significantlygreater in fractures with OP-1 matrix-free devices than the (untreated)fractures at one week postoperative. At two weeks postoperativecontinuing new bone formation was evident in all fractures treated withthe matrix-free OP-1 device. At three weeks, the bone callus was largeand the fractures were substantially or completely healed in thepresence of OP-1. On the left (non-treated) side, however, the fractureline was still evident at three weeks and the amount of callus formedwas less.

The mean torsional load to failure for all ulna treated with any OP-1device was 8.89±2.99N (8) (mean±standard deviation (sample size)). Themean load to failure for non-treated control ulnas was 7.91±2.92N (9).

Greater new bone volume and complete bridging across the fracture sitewas observed in all right (OP-1 device treated) fractures compared tothe left. Proliferation of callus was observed that extended into thesoft tissues of the treated fractures. The left (untreated) sidesuniformly demonstrated new bone proliferation at the periosteal andendosteal borders and early cartilage formation at the fracture, but didnot demonstrate consistent complete bony bridging of the fracture.

Consistent with the radiographic results, greater volume of new bone wasobserved in sites treated with OP-1 devices.

2. Goat Fracture Study

Still another animal model for evaluating enhanced fracture repair usingmatrix-free OP-1 devices is a goat model (tibia midshaft acutefracture). The study compares 0.5 mg of OP-1 in acetate buffer, 1 mgOP-1 in acetate buffer and 1 mg OP-1 precipitated in PBS, injectedimmediately after fracture creation using standard surgical techniques.Animals are followed and cared for as for the dog and rabbit studiesdescribed above and typically are sacrificed at 2, 4 and 6 weeks posttreatment.

It is anticipated that enhanced fracture repair results from inclusionmatrix-free osteogenic devices in these animals as demonstrated for therabbit study.

D. Repair of Osteochondral Defects Using Matrix-Free OP-1 Devices 1.Osteochondral Defects in Rabbits

The following study demonstrates that matrix-free osteogenic devices canenhance repair of both the articular cartilage overlying the bone, aswell as enhancing repair of the underlying bone. In this study, astandard rabbit osteochondral defect model was used to evaluate thevarious injectable forms of OP-1 to heal this kind of defect.

Matrix-free devices containing OP-1 were prepared in two differentinjectable delivery formulations and one freeze-dried formulation. Allsamples contained 125 μg OP-1. Formulation 1: 20 mM acetate buffer, pH4.5 with 5% mannitol, 50 μl full volume; Formulation 2: PhosphateBuffered Saline (PBS) suspension; and Formulation 3: Freeze-dried in 1sample aliquots.

A total of six adult male rabbits were utilized. Full thickness 4.0 mmin diameter osteochondral defects were created bilaterally in thepatellar sulcus of each animal, for a total of 12 defects, usingstandard surgical techniques. The left defect received one of three OP-1formulations and the right side defects acted as an untreated control.All animals were sacrificed at twelve postoperative weeks and the distalfemurs retrieved en bloc. The defect sites were evaluated histologicallyand grossly as described herein above.

In all except one of the PBS group defects, the OP-1 side showssignificant healing with regeneration of both the bone and cartilage.Although healing can be observed in most of the control defects withoutOP-1, the repair is inferior; there is usually incomplete healing of theunderlying bone and a significant underproduction of glycosaminoglycans(GAG) in the cartilage (as seen by light toluidine staining).

2. Sheep Model

Osteochondral and chondral defect repair also can be evaluated in astandard goat or sheep model. For example, using standard surgicaltechniques, each sheep in a study is operated on both foreknee joints,and two defects per joint are created (one each on the medial and thelateral condyle). One of the joints has two standardized partialthickness chondral defects (5 mm in diameter) on each condyle, while theother joint has two similar but deeper full thickness osteochondraldefects (about 1-2 mm in the subchondral bone). One joint animal istreated with a matrix-free osteogenic device formulation, and the otherjoint is left as an untreated control. Each group has a subgroupsacrificed early at 8 weeks and another kept for long term evaluationfor 6-7 months. It is anticipated that matrix-free devices using any ofthe formulations described herein will substantially enhance the speedand quality of repair of both the articular cartilage and the underlyingbone, consistent with the results described herein above.

E. Healing of Non-Critical Size Segmental Defects in Dogs UsingMatrix-Free Osteogenic Devices 1. Experiment 2

As already exemplified in Experiment 1 above (see Section V.A.1.),injectable formulations of rhOP-1 can be used to heal non-critical size(e.g., 5 mm, 3 mm, 1.5 mm) defects. The experiment which follows is anextension of Experiment 1 and focuses on the 3 mm defect model. As isexemplified below in more detail, non-critical size (3 mm) defectstreated with rhOP-1 demonstrated advanced healing and more extensive newbone formation. As demonstrated below, a 3 mm defect provided aconsistent and reproducible model to evaluate acceleration of thefracture repair process.

This experiment evaluates the healing of non-critical size defectstreated with two OP-1 formulations, rhOP-1 in an acetate/lactose buffer(OP/Buffer) and rhOP-1 in a carboxymethylcellulose (CMC) (OP/CMC) gel,at four weeks postoperative. The results summarized below demonstratethat non-critical size defects treated with injectable rhOP-1 in CMCsolultion and in an acetate buffer solution healed significantly fastercompared to CMC and buffer vehicle controls and untreated controls:Radiographically, defects in both OP-1 treatment groups (OP/CMC andOP/Buffer) showed early radiodense bone formation and bridging bone by 4weeks postoperative. The OP/CMC treated defects were almost completelyfilled and spanned with nonuniform density bone along the lateral ulnaborder and incorporating with the host bone cortices. Proliferative newbone was present in the OP/Buffer treated defects. None of the vehiclecontrol defects (CMC and Buffer only) showed evidence of bone defecthealing at 4 weeks. The histologic appearance of OP/CMC and OP/Buffertreated defects was similar. In the OP treated defects, significantamounts of new bone had formed at the defect cortices and along the ulnaperiosteum extending across the defect site. Bone defect bridging wasnearly complete at the 4 week time period. Mineralizing cartilage andfibrous tissue were present in OP treated defects. In contrast, thevehicle control defects were filled and surrounded with fibrous tissueand had minimal amounts of new bone formation at the defect cortices. Onaverage, the OP/CMC treated defects at 4 weeks had a torsional strengththat was 51% of the strength of intact ulnas compared to 14% in the CMCvehicle controls. Defects treated with the OP/Buffer solution had a meantorsional strength that was 44% of intact ulna strength, while thebuffer control defects achieved only 9% of the torsional strength ofintact ulnas. Both the OP/CMC and OP/Buffer treated groups hadmechanical strengths greater than untreated controls at 4 weeks (9%) and8 weeks (27%).

Experimental Design

The test samples consisted of recombinant human osteogenic protein-1(rhOP-1) in an injectable delivery matrix system. Two rhOP-1formulations and two vehicle only controls were evaluated and comparedto previously tested and reported nontreated control defects. Only twoof these three formulations are reported here. One formulation (OP/CMC)consisted of 0.35 mg rhOP-1 in 100 μl carboxymethylcellulose (CMC) gelsupplied in three sterile syringes. A second formulation (OP/Buffer)consisted of 0.35 mg rhOP-1 in 100 μl acetate buffer supplied as an OP-1solution. The vehicle only controls consisted of 100 μl CMC gel (CMCcontrol) supplied in three sterile syringes and 100 μl acetate buffer(Buffer control) supplied as a control solution. Samples of known amountand content were fabricated and supplied sterile by CreativeBioMolecules, Inc. (Hopkinton, Mass.).

Bilateral ulna segmental defects, 3.0 mm in length, were created in allanimals. The right defects received one of two rhOP-1 formulations suchthat three sites of each formulation were studied. The left defectsreceived the vehicle only control containing no rhOP-1. Weeklyradiographs were taken to study the progression of healing. Atsacrifice, all ulnae were retrieved en bloc and if healed sufficiently,mechanically tested in torsion. Segments were evaluated by histology fortissue response, quality and amount of new bone formation and extent ofhealing. Adult male mongrel dogs bred for purpose were utilized in thisstudy because of their availability, ease of handling, anatomical size,and known bone repair and remodeling characteristics. All animals wereskeletally mature, weighed from 44 to 63 pounds (mean 54 lbs), and weresupplied by Martin Creek Kennels, USDA number 71-B-108 (Willowford,Ak.). Special attention was paid in selecting animals of uniform sizeand weight to limit the variability in bone geometry and loading.

Surgery

Anesthesia was administered by intravenous injection of sodium pentothalat the dosage of 5.0 mg/lb body weight. Following induction, anendotracheal tube was placed and anesthesia was maintained byisofluorane inhalation. Both forelimbs were prepped and draped insterile fashion. A lateral incision approximately two centimeters inlength was made and exposure of the ulna was obtained using blunt andsharp dissection. The 3.0 mm sized defect was created in the mid-ulnausing an oscillating saw. The radius was maintained for mechanical studyand no internal or external fixation was used. The site was irrigatedwith saline to remove bone debris and spilled marrow cells. Thesoft-tissues were meticulously closed in layers around the defect. Theappropriate sample formulation was then injected into the defect site asper the treatment schedule. The procedure was then repeated on thecontralateral side with the appropriate sample.

Radiographs

Radiographs of the forelimbs were obtained weekly until four weekspostoperative. Standardized exposure times and intensitites were used.In order to quantify the radiographic results, each radiograph wasassigned a numerical score based on the grading scale described in Table5.

TABLE 5 RADIOGRAPHIC GRADING SCALE Grade No change from immediatepostoperative appearance 0 Trace of radiodense material in defect 1Flocculent radiodensity with flecks of new calcification 2 Defectbridged at least one point with material of non-uniform 3 radiodensityDefect bridged on both medial and lateral sides of defect with 4material of uniform radiodensity, cut end of the cortex remain visibleSame as grade 3; at least one of four cortices is obscured by 5 new boneDefect bridged by uniform new bone; cut ends of cortex are no 6 longerdistinguishable

Sacrifice

Animals were sacrificed using an intravenous barbituate overdose. Theulna and radius were immediately harvested en bloc and placed in salinesoaked diapers. Both ulna were macrophotographed and contact radiographstaken. Soft tissues were carefully dissected away from the defect site.A watercooled saw was used to cult the ulna to a uniform length of 9 cmwith the defect site centered in the middle of the test specimen.

Mechanical Testing

Immediately after sectioning, if healing was deemed sufficient by manualmanipulation, specimens were tested to failure in torsion on an MTSclosed-loop hydraulic test machine (Minneapolis, Minn.) operated instroke control at a constant displacement rate of 50 mm/min. Each end ofthe bone segment was mounted in a cylindrical aluminum sleeve andcemented with methyl methacrylate. One end was rigidly fixed and theother was rotated counterclockwise. Since the dog ulna has a slightcurvature, the specimens were mounted eccentrically to keep specimenrotation coaxial with that of the testing device. The torsional forcewas applied with a lever arm of 6 cm by a servohydraulic materialstesting system. Simultaneous recordings were made of implantdisplacement, as measured by the machine stroke controller, while loadwas recorded from the load cell. Data was recorded via ananalog-to-digital conversion voarch and a personal computer and anonline computer acquisition software. Force angular displacement curveswere generated from which the torque and angular deformation to failurewere obtained, and the energy absorption to failure computed as the areaunder the load-displacement curve.

Histology

Both tested and untested specimens were prepared for histologicevaluation. The individual specimens were fixed by immersion in 10%buffered formalin solution immediately following mechanical testing orafter sectioning in untested specimens. On a water cooled diamond sawthe specimens were divided by bisecting the specimen down its long axis.This procedure resulted in two portions of each specimen for differenthistologic preparations including undecalcified ground sections andundecalcified microtome sections.

Following fixation, the specimens designated for undecalcified sectionswere hydrated in graduated ethyl alcohol solutions from 70% to 100%. Thespecimens were then placed in methyl methacrylate monomer and allowed topolymerize. The ground sections were obtained by cutting the specimenson a high speed, water cooled Mark V CS600-A (East Grandy, Conn.)sectioning saw into sections approximately 700 to 1,000 microns thick.These sections were mounted on acrylic slides and ground to 100 micronthickness using a metallurgical grinding wheel, and microradiographswere made using standardized techniques. Following microradiography thesections were further ground to approximately 50 microns and stainedwith basic fuchsin and toluidine blue for histologic grading thatevaluated the quality of the union, the appearance and quality of thecortical and cancellous bone, the presence of bone marrow elements, boneremodeling, and inflammatory response (Table 6).

TABLE 6 HISTOLOGIC GRADING SCALE Grade Quality of Union No sign offibrous of other union 0 fibrous union 1 osteochondral union 2 boneunion 3 bone union with reorganization of 4 cortices Cortex Developmentnone present in the defect 0 densification of borders 1 recognizableformation 2 intact cortices but not complete 3 complete formation ofnormal 4 cortices Inflammatory Response severe response 0severe/moderate response 1 moderate response 2 mild response 3 noresponse 4 TOTAL POINTS 12 

EXPERIMENTAL RESULTS Radiographic Evaluation

A summary of the radiographic grades for each site is provided in Table7. At 4 weeks postoperative, defects treated with OP/CMC had a meanradiographic grade of 3.0 out of 6 possible points. Defects treated withOP/Buffer had a mean radiographic grade of 4.0. Defects treated with CMCvehicle control and buffer only control averaged final radiographicgrades of 1.33 and 1.0, respectively. In both OP-1 treated groups,OP/CMC and OP/Buffer, there were signs of radiodense new bone forming inthe defects and along the lateral defect borders as early as three weekspostoperative. At four weeks, significant amounts of new bone had formedwithin the defects and in surrounding subcutaneous tissue. The OP/CMCdefects were almost completely filled and spanned with nonuniformdensity bone along the lateral ulna border. New bone was significantlyincorporated with the defect cortices. In two of three OP/Buffer treateddefects, the host cortices remained visible although proliferative newbone was present. In contrast, none of the OP/CMC or OP/Buffer defectswere completely bridged or filled by four weeks postoperative. In theCMC control group, early new bone obscured the host bone cortices atthree weeks and continued to increase in radiodensity. Again, incontrast, the buffer control defects showed only a slight increase inradiodensity at the defect cortices at four weeks. None of the controldefects in either group showed evidence of bony defect healing.

TABLE 7 RADIOGRAPHIC GRADING RESULTS Implant Type 1 week 2 weeks 3 weeks4 weeks OP/CMC 0 1 2 3 OP/CMC o o 1 3 OP/CMC o 1 2 3 CMC Control o o 1 1CMC Control o 1 1 2 CMC Control o o o 1 OP/Buffer o 1 2 3 OP/Buffer o 12 4 OP/Buffer o 1 2 5 Buffer control o o o 1 Buffer control o o o 1Buffer control o o 1 1 OP/CMC 0.0 ± 0.0 0.67 ± 0.58 1.67 ± 0.58 3.0 ±0.0 mean ± st dev (n) (3) (3) (3) (3) CMC Control 0.0 ± 0.0 0.33 ± 0.580.67 ± 0.58 1.33 ± 0.58 mean ± st dev (n) (3) (3) (3) (3) OP/Buffer 0.0± 0.0 1.0 ± 0.0 2.0 ± 0.0 4.0 ± 1.0 mean ± st dev (n) (3) (3) (3) (3)Buffer control 0.0 ± 0.0 0.0 ± 0.0 0.33 ± 0.58 1.0 ± 0.0 mean ± st dev(n) (3) (3) (3) (3) OP/CMC = 0.35 mg rhOP-1 in 100 μl CMC gel CMCControl = 100 μl CMC vehicle only gel OP/Buffer = 0.35 mg rhOP-1 in 100μl acetate buffer solution Buffer control = 100 μl acetate buffervehicle only solution

OP/CMC—4 weeks

At one week postoperative, there were no changes in radiographicappearance of any OP/CMC defects. At 2 weeks, trace radiodense areaswere present at the cut bone ends. At 3 weeks, there was an increase inradiodensity of new bone forming within the defects and along thelateral defect borders. One defect showed signs of early bony bridging.At 4 weeks, the OP/CMC defects had a significant amount of radiodensenew bone both within the defect. The defects were almost completelyfilled and spanned with nonuniform density bone along the lateral ulnaborder. New bone was significantly incorpoated with the defect cortices.None of the OP/CMC treated defects were completely filled or solidlybridged with new bone at 4 weeks postoperative. The final radiographicgrade for each defect was 3 out of 6 possible points (mean 3.0±0.0,n=3).

CMC Control—4 weeks

At two weeks postoperative, there were no significant changes inradiographic appearance of any CMC control defects. At 3 weeks, the hostcortices were beginning to obscure with new bone in 2 of 3 defects. At 4weeks, the CMC defects showed some evidence of new bone activity at thedefect cortices but no evidence of bony defect healing. The finalradiographic grades were 1, 2 and 1 out of 6 possible points (mean1.33±0.58, n=3).

OP/Buffer—4 weeks

At one week postoperative, there were no changes in radiographicappearance of any OP/Buffer treated defect. At two weeks postoperative,trace radiodense areas were present within the OP/buffer defects andalong the defect borders. Significant new bone formation was also seenin the subcutaneous tissues surrounding the defects. At 3 weeks, flecksof new bone appeared in the defects and new bone formed in the overlyingsoft tissues. At 4 weeks, there was a significant increase in radiodensenew bone formation filling and bridging the OP-1 treated defects. In twoof three defects the cortices remained visible although proliferativenew bone filled and spanned the defects. None of the OP/Buffer treateddefects were completely filled or solidly bridged with new bone at thesacrifice period of 4 weeks. The final radiographic grades were 3,4 and5 out of 6 possible points, respectively (mean 4.0±1.0, n=3).

Buffer Control—4 weeks

At 3 weeks postoperative, there were no significant changes inradiographic appearance of any defect treated with the buffer onlycontrol. At 4 weeks, an increase in radiodensity at the cortices wasobserved although no signs of defect healing were evident. The finalradiographic grade for each site was 1 out of 6 possible points (mean1.0±0.0, n=3).

Gross Observations

OP-1 defects: All OP/CMC and OP/Buffer treated defects were manuallystable and visibly had a mass of new bone formation at the defect site.Vehicle control defects: None of the CMC and Buffer only control defectswere manually stable at 4 weeks although all were mechanically tested.

Mechanical Testing

A summary of the mechanical testing results appears in Table 8.

OP/CMC

At 4 weeks postoperative, the mean load to failure of 3 mm defectstreated with OP/CMC was 33.08±16.41N (n=3). This represented 51% of thestrength of intact controls tested previously. The mean angulardeformation was 31.13±15.32 degrees. The mean energy absorbed to failurewas 41.64±30.52 Nm-degrees.

CMC Control

At 4 weeks postoperative, the mean load to failure of 3 mm defectstreated with CMC control was 9.32±16.41N (n=3). This represented 14% ofthe strength of intact controls tested previously. The mean angulardeformation was 33.36±25.95 degrees. The mean energy absorbed to failurewas 10.53±8.62 Nm-degrees.

OP/Buffer

At 4 weeks postoperative, the mean load to failure of 3 mm defectstreated with OP/Buffer was 29.03±16.79N (n=3). This represented 44% ofthe strength of intact controls tested previously. The mean angulardeformation was 36.14±14.71 degrees. The mean energy absorbed to failurewas 37.87±27.73 Nm-degrees.

Buffer Control

At 4 weeks postoperative, the mean load to failure of 3 mm defectstreated with Buffer control was 5.62±1.65N (n=3). This represented 9% ofthe strength of intact controls tested previously. The mean angulardeformation was 24.91±12.03 degrees. The mean energy absorbed to failurewas 3.94±4.12 Nm-degrees.

TABLE 8 MECHANICAL TESTING RESULTS Energy Maximum Percent absorbed toLoad to intact failure Failure Torque control Angul- (Nm- Implant (N)(Nm) (%) ation degrees) OP/CMC 49.37 2.96 75.65 35.72 63.57 OP/CMC 16.560.99 25.37 14.04 6.78 OP/CMC 33.32 2.00 51.05 43.62 54.56 MEAN ± 33.08±1.99± 50.69± 31.13± 41.64± STANDARD 16.41 0.98 25.14 15.32 30.52DEVIATION CMC Control 12.81 0.77 19.63 33.26 14.06 CMC Control 11.008.00 16.85 59.35 16.83 CMC Control 4.14 0.25 6.34 7.46 0.70 MEAN ± 9.32±3.01± 14.27± 33.36± 10.53± STANDARD 4.57 4.33 7.01 25.95 8.62 DEVIATIONOP/Buffer 32.47 1.95 49.75 50.11 55.91 OP/Buffer 43.83 2.63 67.15 37.5351.77 OP/Buffer 10.79 0.65 16.53 20.78 5.94 MEAN ± 20.03± 1.74± 44.48±36.14± 37.87± STANDARD 16.79 1.01 25.72 14.71 27.73 DEVIATION Buffercontrol 4.82 0.29 7.38 11.12 0.73 Buffer control 4.53 0.27 6.94 30.322.50 Buffer control 7.52 0.45 11.52 33.29 8.59 MEAN ± 5.62± 0.34± 8.62±24.91± 3.94± STANDARD 1.65 0.10 2.53 12.03 4.12 DEVIATION

Histology

A summary of the histologic grading results appears in Table 9. Out of12 total points, the mean histologic grade of defects treated withOP/CMC was 7.00±0.87. The mean histologic grade of the CMC controldefects was 4.50±0.87. The mean histologic grades of the OP/Bufferdefects and the Buffer controls were 6.08±0.14 and 4.0±1.0,respectively.

OP/CMC

Treatment resulted in early osteochondral bridging with areas ofmineralizing cartilage at four weeks. In defects treated with OP/CMC,significant new bone formation was observed in the periosteal andendosteal regions of the ulna and extended beyond the defect borders.Areas of mineralizing cartilage and some fibrous tissue were presentwithin the defects. Bridging of the defects was not complete by fourweeks. The host cortices remained visible although there were signs ofnew bone incorporation and remodelling.

CMC control

No complete bony healing was observed in any CMC control defects at fourweeks. Control defects resulted in fibrous unions with no signs of bonybridging. Fibrous tissue and mineralizing cartilage was observed fillingand surrounding the defects. Very small amounts of new bone had formedalong the ulna periosteum and at the endosteal region of the ulna nearthe host cortices. Signs of host cortex resorption were observed at thedefect ends.

OP/Buffer

Treated defects were filled with mineralizing cartilage and fibroustissue. New bone formed in the endosteal and periosteal regions of theulna near the defect borders and early signs of bridging with new bonewas evident althoug none of the defects were completely spanned. Thehost bone cortices showed signs of incorporation with new bone but werenot completely obscured by four weeks. Some remodelling of the host bonecortices and early densification along the new bone borders wasobserved. New bone also formed in the subcutaneous tissue layersoverlying the defect site and extended beyond the defect borders.

Buffer control

No complete bony healing was observed in any buffer control defects atfour weeks postoperative. Untreated defects exhibited fibrous unionswith no signs of bony bridging; fibrous tissue was observed filling andsurrounding the defects. Other untreated defects showed no sign offibrous or other union. Very little new bone formation was observed inthe buffer control defects. Endosteal new bone extended from the ulnamarrow cavity and periosteal new bone formed along the lateral defectborders. The host bone ends were visible with signs of corticalresorption.

TABLE 9 HISTOLOGIC GRADING RESULTS Infamm- Quality Cortex atory TotalImplant of Union Development Response Score OP/CMC 1.5 1 4 6.5 OP/CMC 31 4 8 OP/CMC 1.5 1 4 6.5 MEAN ± 2.0 ± 87  1.0 ± 0.0 4.0 ± 0.0  7.0 ±0.87 STANDARD DEVIATION CMC Control 1 0 4 5 CMC Control 1 0 2.5 3.5 CMCControl 1 0 4 5 MEAN ± 1.0 ± 0.0 0.0 ± 0.0 3.50 ± 0.87 4.50 ± 0.87STANDARD DEVIATION OP/Buffer 1.25 1 4 6.25 OP/Buffer 1 1 4 6 OP/Buffer 11 4 6 MEAN ± 1.08 ± 0.14 1.0 ± 0.0 4.0 ± 0.0 6.08 ± 0.14 STANDARDDEVIATION Buffer control 1 0 2 3 Buffer control 1 0 4 5 Buffer control 00 4 4 MEAN ± 0.67 ± 0.58 0.0 ± 0.0 3.33 ± 1.15 4.0 ± 1.0 STANDARDDEVIATION

2. Experiment 3

Recombinant human osteogenic protein-1 (rhOP-1), when implanted incombination with bone collagen matrix, has been shown to healcritical-sized diaphyseal segmental defects in animals with theformation of new bone that is both biologically and biomechanicallyfunctional. The purpose of this study was to evaluate the efficacy ofmatrix-free injectable formulations of rhOP-1 for accelerating bonehealing in a canine non-critical-sized defect model.

Bilateral osteoperiosteal segmental defects, 3.0 mm in length, werecreated in the mid-ulna of 18 adult male mongrel dogs. The radius wasmaintained for mechanical stability without additional fixation. Softtissues were closed prior to injection of rhOP-1. Nine animals receivedrhOP-1 formulations in one defect and vehicle controls in thecontralateral defect and were sacrificed at 4 weeks postoperative. Nineuntreated control defects were evaluated at periods of 4, 8 and 12 weeksfor comparison with the rhOP-1 treatment. Radiographs were taken atregular intervals to study the progression of healing. At sacrifice, allulnae were mechanically tested in torsion if healing was sufficient.Undecalcified histologic sections were evaluated for quality and amountof new bone formation and extent of healing.

Radiographically, new bone formation was evident as early as two weekspostoperative in rhOP-1 treated defects and at 4 weeks, new bone bridgedthe defect. In contrast, vehicle control sites showed little or no boneformation at 4 weeks postoperative. Moreover, torsional strengths ofdefects treated with rhOP-1 were significantly greater at 4 weeks thanvehicle or untreated controls at 4 weeks. Furthermore, torsionalstrength of treated defects at 4 weeks virtually equaled the strength ofuntreated controls at 12 weeks. A clear acceleration of defect healingand bone formation resulted from rhOP-1 treatment. Histologic findingscorrelated with radiographic and mechanical testing results.

The results of this study confirm that osteogenic proteins injected innon-critical-sized defects can accelerate defect healing, includingfracture callus formation and bridging bone formation. Defects treatedwith rhOP-1 formed new bone significantly faster and restored fracturestrength and stiffness earlier than untreated controls.

In summary, the ability of the matrix-free devices described hereinaboveto substantially enhance defect repair, including accelerating the rateand enhancing the quality of newly formed bone, has implications forimproving bone healing in compromised individuals such as diabetics,smokers, obese individuals, aged individuals, individuals afflicted withosteoporosis, steroidal users and others who, due to an acquired orcongenital condition, have a reduced capacity to heal bone fractures,including individuals with impaired blood flow to their extremities.Such individuals experience refractory healing, resulting from a reducedcapacity to promote progenitor cells, and are subject to gangrene and/orsepsis.

The methods and formulations disclosed herein provide enhanced bonerepair by accelerating bone formation. Specifically, following themethods and protocols disclosed herein, the rate of bone formation,including bone callus formation and bridging can be accelerated. Asexemplified herein, bridge formation occurs faster, and in a shortertime frame, allowing for more stable bone formation, thereby enhancingbiomechanical strength of the newly forming bone.

It is well-known in the art that callus formation is one stage in themulti-staged ealing process culminating in bone formation. Specifically,the healing process involves five stages: impact, inflammation, softcallus formation, hard callus formation, and remodeling. Impact beginswith the initiation of the fracture and continues until energy hascompletely dissipated. The inflammation stage is characterized byhematoma formation at the fracture site, bone necrosis at the ends ofthe fragments, and an inflammatory infiltrate. Granulation tissuegradually replaces the hematoma, fibroblasts produce collagen, andosteoclasts begin to remove necrotic bone. The subsidence of pain andswelling marks the initiation of the third, or soft callus, stage. Thisstage is characterized by increased vascularity and abundant newcartilage formation. The end of the soft callus stage is associated withfibrous or cartilaginous tissue uniting the fragments. During thefourth, or hard callus, stage, the callus converts to woven bone andappears clinically healed. The final stage of the healing processinvolves slow remodeling from woven to lamellar bone and reconstructionof the medullary canal (see “Current Diagnosis & Treatment inOrthopedics,” ed. H. B. Skinner (LANGE Medical Book Publ.)).

F. Repair of Chondral Defects with Matrix-Free Osteogenic Devices(Sheep) 1. Experiment 1

Using materials and methods similar to those described above (seerelevant portions of D.2), the following study was conducted to furtherdemonstrate that the exemplary osteogenic protein OP-1, whenadministered in a matrix-free device, can induce active chondrogenesisand chondral defect repair in weight-bearing joints.

As already described above, a defect is a structural disruption of thecartilage and can assume the configuration of a void, athree-dimensional defect such as, for example, a gap, cavity, hole orother substantial disruption in structural integrity. Defects inarticular cartilage may extend through the entire depth of articularcartilage and/or into the subchondral bone (osteochondral defects) ordefects may be superficial and restricted to the cartilage tissue itself(chondral or subchondral defects).

Initially, damaged cartilage matrix undergoes degradation bymetalloproteinases that are released by nearby cellular constituents.Proteolytic degradation clears damaged matrix components therebyreleasing anabolic cytokines entrapped in the matrix. As currentlyunderstood, cytokines released from the matrix stimulate proliferationof chondrocytes and, importantly, synthesis of a new macromolecularmatrix. The presence of clusters of proliferating chondrocytes, asdetermined microscopically, is one of the first indicators of acartilage reparative response. Presumably, this repair response countersthe catabolic effect of proteases and stabilizes the tissue by enhancedmatrix synthesis.

Articular cartilage and repair of articular cartilage is readily studiedby standard histological and histochemical means. The techniques arewell-known in the art and include microscopic examination of sections ofcartilage stained by any one of a number of histochemical stainsincluding, but not limited to, toluidine blue, hematoxylin and eosin,von Kossa, safranin O, and Masson's trichrome stain. Following theapplication of different stains, the skilled artisan can assess thereparative response of cartilage by identification of proliferatingchondrocytes and determination of the quality and quantity of matrix,such as collagen and proteoglycans, synthesized by chondrocytes.

As used herein, articular cartilage refers specifically to hyalinecartilage, an avascular, non-mineralized tissue which covers thearticulating surfaces of the portions of bones in joints. Underphysiological conditions, articular cartilage overlies highly vascularmineralized bone called subchondral bone. Articular cartilage ischaracterized by specialized cartilage forming cells, calledchondrocytes, embedded in an extracellular matrix comprising fibrils ofcollagen (predominantly Type II collagen as well as the minor types suchas Types IX and XI), various proteoglycans, includingglycosaminoglycans, other proteins and water.

In this study, sheep were used as a model to assess repair of 1-2 mmtotal depth×7 mm total diameter chondral defects on the weight-bearingcondylar surface of the knee. The defects were partial thicknesschondral defects and did not involve the subchondral bone as was evidentby a lack of bleeding following defect creation. Further confirmationwas obtained by histology of thin sections at the time of sacrifice; thedefects did not extend into subchondral bone.

The experimental protocol is provided in Table 10. Using standardizedsurgical techniques, a 2 mm total depth×7 mm total diameter defect wassurgically made on the weight-bearing surface of each condyle of theright and left knee. The right knee served as the control knee. A liquidmatrix-free OP-1 device (50 or 250 μg OP-1) in 20 mM sodium acetate, pH4.5, was delivered either as a single bolus via injection into theintra-articular joint, or intermittently delivered (0.5 μL per hour fora 2 week duration; 200 μL total) via a locally implanted, subcutaneousmini-pump (ALZET® 2002, ALZA Scientific Products, Palo Alto, Calif.).Numerous suitable mini-pumps are readily available and routinely used bythe skilled practitioner for delivery of pharmaceuticals and/orherapeutic agents; the skilled artisan will appreciate the preferredmode and rate of delivery under the circumstances. Healing of chondraldefects was assessed by standard histological and histochemical methods.

TABLE 10 Chondral Defect Repair in Sheep Group Left Knee (Matrix-freeDevice) Right Knee (Cntrl) I  50 μg OP-1 No Rx II 250 μg OP-1 No Rx III 50 μg OP-1 via mini-pump Vehicle via mini-pump IV 250 μg OP-1 viamini-pump Vehicle via mini-pump

The results collected to date of a 3 month mini-pump study (Group IIIand IV) reveal that matrix-free OP-1 devices can induce chondrogenesisand subsequent repair of chondral defects. Little evidence of chondraldefect repair was observed at 12 weeks in the control defects. However,by standard histological and histochemical evaluation, new cartilageformation as well as fusion of the old and new cartilage was found inthe matrix-free OP-1 treated animal. Using art-recognized histologicaland histochemical indicia as a measure of chondral repair, OP-1stimulated the ingrowth of synovial cells into the defect area. Thesecells differentiated into full thickness, proteoglycan-rich articularchondrocytes and repair of the chondral defect resulted therefrom.

The healing of a partial thickness cartilage defect without subchondralbone involvement in an adult animal is unprecedented and demonstratesthat active chondrogenesis is a feature of the repair process that isinduced by a matrix-free osteogenic device. It is concluded from thesestudies that a matrix-free osteogenic device can be used to repairchondral defects in vivo. It is particularly significant that suchrepair can occur at a weight-bearing joint in a large animal model suchas the sheep.

Other studies of chondral defect repair using matrix-free OP-1 devices(for example, experiments as outlined in Group I and II above) arecurrently still in progress. Results similar to those obtained with themini-pump delivery experimental paradigm described above are expected,that is, a single bolus of injectable matrix-free device injected intothe intra-articular joint is expected to repair chondral defects inweight-bearing joints.

G. Alternative Methods of Healing Non-Critical Size Segmental DefectsUsing Matrix-Free Osteogenic Devices 1. Experiment 1: The Effects ofDelayed Administration of Matrix-Free Osteogenic Device on Repair ofNon-Critical Size Defects (Dogs)

The purpose of this study was to evaluate the healing of non-criticalsize defects treated with matrix-free OP-1 devices at various delayedadministration times post-injury. The particular device exemplifiedbelow is an injectable formulation of the matrix-free osteogenic device.Other device embodiments are expected to yield similar results.

Briefly, the experimental observations are as follows: In general,non-critical size segmental defects treated with matrix-free OP-1devices healed to a significantly greater degree compared to injectablecarrier controls at 4 weeks post-injury. Of particular significance arethe unexpected results which indicate that at least one indicia ofdefect healing, specifically, enhanced ulnar mechanical strength, can beenhanced by manipulating the post-injury time at which matrix-free OP-1devices are administered.

For purposes of this experiment and as used herein, injury meansaccidental occurrence of a defect (such as an unexpected physical mishapresulting in the occurrence of a non-critical size defect), purposefuloccurrence of a defect (such as surgical manipulation resulting in theoccurrence of a non-critical size defect), or non-traumatically induceddefects caused by one or more of the following diseases or disorders:hypoxia; ischemia; primary and metastatic neoplasia; infectiousdiseases; inflammatory diseases; so-called collagen diseases (includingdefects in collagen synthesis, degradation or normal matrix);congenital, genetic or developmental diseases;

nutritional diseases; metabolic diseases; idiopathic diseases; anddiseases associated with abnormal mineral homeostasis, to name but afew. Certain of the methods exemplified herein contemplate the step ofadministering a matrix-free device to a defect site after onset of thehealing process; the stages of the healing process, and thephysiological events associated therewith, were earlier described.Another of the methods of the present invention comprises the step ofadministering a matrix-free device to a defect site during maturation ofthe endogenous matrix at the site; the events associated with endogenousmatrix formation during endochondral bone formation were also earlierdescribed. In a currently preferred embodiment, the present inventionprovides a method of repairing a bone defect, chondral defect, orosteochondral defect involving the step of administering a matrix-freedevice at times post-injury which are delayed. Such delays can beshort-term, moderate or long-term as described below. The extent towhich administration is delayed depends upon the circumstances and theskilled artisan will readily appreciate the significance thereof.

As demonstrated below, improved healing and defect repair results fromadministration of a matrix-free device to a defect site at elapsed timespost-injury. For example, delayed administration times can include timesfrom at least 0.5 hours to at least 6.0 hours post-injury;alternatively, delayed administration times can include times from atleast 6 hours to 24 hours or from at least 24 hours to 48 hourspost-injury. In one currently preferred embodiment, the delay is atleast 6 hours. Other post-injury administration times are alsocontemplated by the instant invention. In certain other currentlypreferred embodiments, delayed administration times can range from atleast 48 hours to at least 72 hours post-injury. In yet otherembodiments, administration times can range significantly beyond 72hours, e.g., matrix-free osteogenic devices can be administered to thedefect site as late as the remodeling stage of bone healing. Alsocontemplated are methods wherein matrix-free osteogenic devices areadministered to a non-critical size defect site at a plurality of timepoints post-injury. For example, a currently preferred plurality is from0.5 to 6 hours and 7 days post-injury. A plurality of delayedadministrations can be accomplished by manual delivery to the defectlocus or by automated delivery using a mini-pump as described earlier.

Experimental Design

A total of 12 adult mongrel dogs were utilized. As described earlier,bilateral ulnar segmental defects, 3.0 mm in length, were created in allanimals. As exemplified in this particular study, the matrix-freeformulation of OP-1 used was 3.5 mg OP-1/ml delivered in 100 microLlactose/acetate buffer as described earlier. Twelve animals wereadministered matrix-free devices in the right defect at variouspost-injury time points and control devices were administered in theleft defect at various post-injury time points. Three animals weretreated at defect creation (0 hours), three at 6 hours post-injury, andthree at 48 hours post-injury. All animals were sacrificed 4 weeks aftersurgery. Weekly radiographs were taken to study the progression ofhealing. At sacrifice, segments of bone were evaluated by histology fortissue response, quality and amount of new bone formation, and extent ofhealing. All ulnae were retrieved en bloc and mechanically tested intorsion.

As described earlier, immediately after sectioning, ulna were tested tofailure in torsion on an MTS closed-loop hydraulic test machine operatedin stroke control at a constant displacement rate of 50 mm/min. One endwas rigidly fixed and the other was rotated counterclockwise. Thetorsional force was applied with a lever arm of six cm, by aservohydraulic materials testing system. Simultaneous recordings weremade of implant displacement, as measured by the machine strokecontroller, while load was recorded from the load cell. Data wasrecorded via an analog-to-digital conversion board and a personalcomputer and on-line computer acquisition software. Force-angulardisplacement curves were generated from which the torque and angulardeformation to failure were obtained, and the energy absorption tofailure computed as the area under the load-displacement curve.

Results

All specimens were mechanically tested at 4 weeks post-surgery.Mechanically, defects receiving matrix-free OP-1 devices at 6 hourspost-injury had the highest torsional strength; 73% of intact ulnaecompared to 64% at 48 hours and 60% at 0 hours. The control defects at 0hours, 6 hours, and 48 hours post-injury had strengths of 23%, 28%, and24%, respectively.

This study demonstrates the surprising result that, in certainembodiments of the present invention, improved healing of a non-criticalsize defect can be achieved by delayed administration of a matrix-freeosteogenic device to the defect locus post-injury. This unexpectedresult is related to the stage of bone healing or endogenous matrixformation at the defect site including but not limited to events such asclot formation, progenitor cell infiltration, and callus formation,particularly soft callus, to name but a few. Moreover, it is expectedthat other defect repair processes involving bone, such as repair ofosteochondral defects similar to those described herein, can be improvedby delayed administration of matrix-free osteogenic devices to thedefect site post-injury.

H. Further Studies of Chondral Defect Repair Using Matrix-FreeOsteogenic Devices (Sheep) 1. Experiment 1: Glycosaminoglycans and OtherPolymers as a Carrier for Osteogenic Protein

As described earlier, certain preferred categories of compounds aresuitable as carriers in the matrix-free devices contemplated herein.Among the currently preferred categories are compounds appreciated bythe art as lubricating agents, especially those which occur naturallyand naturally perform physiological functions such as protection andlubrication of cells and maintenance of tissue integrity, to name but afew. Such compounds are generally also wetting and moisture-preservingagents. One sub-category of currently preferred lubricating agentsincludes the biopolymers known as glycosarninoglycans.Glycosaminoglycans contemplated by the present invention include, butare not limited to, hyaluronic acid, chondroitin, dermatan, keratan toname but a few. Sulfonated as well as non-sulfonated forms can be usedin the present invention. Other glycosaminoglycans are suitable forformulating matrix-free devices, and those skilled in the art willeither know or be able to ascertain other suitable compounds using nomore than routine experimentation. For a more detailed description ofglycosaminoglycans, see Aspinall, Polysaccharides, Pergamon Press,Oxford (1970).

A particularly preferred glycosaminoglycan is hyaluronic acid (HA). HAis a naturally occurring anionic polysaccharide or complex sugar. It isfound in cartilage and synovial fluid. HA is available both in cosmeticgrade and medical grade; medical grade is generally preferred for usewith the present invention. HA can range in molecular weight from low tohigh. In certain embodiments of the present invention, high molecularweight material is preferable; as an example only, HA 190 (1.9 a 10⁶ Da;1% is currently preferred, yet concentrations ranging from 0.5-2.0% aresuitable) admixed with saline can be administered (0.1 ml/kg) twiceweekly intra-articularly to repair chondral defects. In otherembodiments, low molecular weight HA (such as HA80, 0.8×10⁶ Da; 1% ispreferred, yet concentrations less than or equal to 4% are suitable) canbe used. Using the teachings provided herein, the skilled artisan canassess the circumstances under which high molecular weight HA ispreferable to low molecular weight material for defect repair, and viceversa. Moreover, the skilled artisan will further appreciate that HA insolution is a viscous liquid and that the viscosity can be manipulatedby adjusting the molecular weight and the HA content. For example, insome embodiments, it is preferred to approximate the viscosity ofsynovial fluid in the joint. Using ordinary skill and routineexperimentation, together with the teachings provided herein, theskilled artisan can formulate matrix-free osteogenic devices using HA asa carrier for chondral defect repair; the viscosity of the device aswell as the protein content can be readily adjusted as required by thecircumstances and as taught herein. HA is available commercially fromseveral sources including Sigma Chemical Company (St. Louis, Mo.),Genzyme Pharmaceuticals (Cambridge, Mass.) and CollaborativeLaboratories (East Setauket, N.Y.).

Glycosaminoglycan and other polymeric carriers, such as hyaluronic acid,suitable for use with the instant matrix-free osteogenic devices can beevaluated in the sheep chondral defect model described above. Forexample, two 2×7 mm defects are made by standard surgical procedures inthe weight bearing surface of the medial and lateral condyles of bothknee joints in a sheep. One knee joint is treated by intra-articularadministration of an OP-1/hyaluronic acid matrix-free device and theother joint is treated with hyaluronic acid alone.

Two groups of sheep are studied: Group I is sacrificed at 8 weeks andGroup II is sacrificed at 6 months. As described earlier, healing ofchondral defects can be assessed by radiology and standard histologicaland histochemical methods. Radiographs of each knee are taken at monthlyintervals. Arthroscopic examination is performed using standardtechniques and equipment under anesthesia just prior to sacrifice.Immediately after sacrifice, specimens of the knee joint are fixed in10% neutral buffered formalin. Specimens are bisected longitudinally andone section decalcified in graduated ethyl alcohol solutions from70-100%, and embedded in methylmethacrylate, sectioned and stained forhistologic grading.

It is expected that hyaluronic acid-containing matrix free devices canenhance the rate of chondral defect repair and can improve the extent ofrepair achieved. Repair can be assessed in animal models by standardcartilage characterization methods, including histologic grading ofstained and fixed tissue sections, localization of cartilage-specificmacromolecules (such as type II collagen and aggrecan), determination ofthe proteoglycan profile and mechanical testing. All the foregoing canbe accomplished by the skilled artisan using routine experimentation andthe knowledge in the art.

Equivalents

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting on the invention described herein. Scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes which come within the meaning andrange of equivalency of the claims are therefore intended to be embracedtherein.

8 1822 base pairs nucleic acid single linear cDNA No No HOMO SAPIENSHIPPOCAMPUS CDS 49..1341 experimental /function= “OSTEOGENIC PROTEIN”/product= “OP1” /evidence= EXPERIMENTAL /standard_name= “OP1” 1ggtgcgggcc cggagcccgg agcccgggta gcgcgtagag ccggcgcg atg cac gtg 57 MetHis Val 1 cgc tca ctg cga gct gcg gcg ccg cac agc ttc gtg gcg ctc tgggca 105 Arg Ser Leu Arg Ala Ala Ala Pro His Ser Phe Val Ala Leu Trp Ala5 10 15 ccc ctg ttc ctg ctg cgc tcc gcc ctg gcc gac ttc agc ctg gac aac153 Pro Leu Phe Leu Leu Arg Ser Ala Leu Ala Asp Phe Ser Leu Asp Asn 2025 30 35 gag gtg cac tcg agc ttc atc cac cgg cgc ctc cgc agc cag gag cgg201 Glu Val His Ser Ser Phe Ile His Arg Arg Leu Arg Ser Gln Glu Arg 4045 50 cgg gag atg cag cgc gag atc ctc tcc att ttg ggc ttg ccc cac cgc249 Arg Glu Met Gln Arg Glu Ile Leu Ser Ile Leu Gly Leu Pro His Arg 5560 65 ccg cgc ccg cac ctc cag ggc aag cac aac tcg gca ccc atg ttc atg297 Pro Arg Pro His Leu Gln Gly Lys His Asn Ser Ala Pro Met Phe Met 7075 80 ctg gac ctg tac aac gcc atg gcg gtg gag gag ggc ggc ggg ccc ggc345 Leu Asp Leu Tyr Asn Ala Met Ala Val Glu Glu Gly Gly Gly Pro Gly 8590 95 ggc cag ggc ttc tcc tac ccc tac aag gcc gtc ttc agt acc cag ggc393 Gly Gln Gly Phe Ser Tyr Pro Tyr Lys Ala Val Phe Ser Thr Gln Gly 100105 110 115 ccc cct ctg gcc agc ctg caa gat agc cat ttc ctc acc gac gccgac 441 Pro Pro Leu Ala Ser Leu Gln Asp Ser His Phe Leu Thr Asp Ala Asp120 125 130 atg gtc atg agc ttc gtc aac ctc gtg gaa cat gac aag gaa ttcttc 489 Met Val Met Ser Phe Val Asn Leu Val Glu His Asp Lys Glu Phe Phe135 140 145 cac cca cgc tac cac cat cga gag ttc cgg ttt gat ctt tcc aagatc 537 His Pro Arg Tyr His His Arg Glu Phe Arg Phe Asp Leu Ser Lys Ile150 155 160 cca gaa ggg gaa gct gtc acg gca gcc gaa ttc cgg atc tac aaggac 585 Pro Glu Gly Glu Ala Val Thr Ala Ala Glu Phe Arg Ile Tyr Lys Asp165 170 175 tac atc cgg gaa cgc ttc gac aat gag acg ttc cgg atc agc gtttat 633 Tyr Ile Arg Glu Arg Phe Asp Asn Glu Thr Phe Arg Ile Ser Val Tyr180 185 190 195 cag gtg ctc cag gag cac ttg ggc agg gaa tcg gat ctc ttcctg ctc 681 Gln Val Leu Gln Glu His Leu Gly Arg Glu Ser Asp Leu Phe LeuLeu 200 205 210 gac agc cgt acc ctc tgg gcc tcg gag gag ggc tgg ctg gtgttt gac 729 Asp Ser Arg Thr Leu Trp Ala Ser Glu Glu Gly Trp Leu Val PheAsp 215 220 225 atc aca gcc acc agc aac cac tgg gtg gtc aat ccg cgg cacaac ctg 777 Ile Thr Ala Thr Ser Asn His Trp Val Val Asn Pro Arg His AsnLeu 230 235 240 ggc ctg cag ctc tcg gtg gag acg ctg gat ggg cag agc atcaac ccc 825 Gly Leu Gln Leu Ser Val Glu Thr Leu Asp Gly Gln Ser Ile AsnPro 245 250 255 aag ttg gcg ggc ctg att ggg cgg cac ggg ccc cag aac aagcag ccc 873 Lys Leu Ala Gly Leu Ile Gly Arg His Gly Pro Gln Asn Lys GlnPro 260 265 270 275 ttc atg gtg gct ttc ttc aag gcc acg gag gtc cac ttccgc agc atc 921 Phe Met Val Ala Phe Phe Lys Ala Thr Glu Val His Phe ArgSer Ile 280 285 290 cgg tcc acg ggg agc aaa cag cgc agc cag aac cgc tccaag acg ccc 969 Arg Ser Thr Gly Ser Lys Gln Arg Ser Gln Asn Arg Ser LysThr Pro 295 300 305 aag aac cag gaa gcc ctg cgg atg gcc aac gtg gca gagaac agc agc 1017 Lys Asn Gln Glu Ala Leu Arg Met Ala Asn Val Ala Glu AsnSer Ser 310 315 320 agc gac cag agg cag gcc tgt aag aag cac gag ctg tatgtc agc ttc 1065 Ser Asp Gln Arg Gln Ala Cys Lys Lys His Glu Leu Tyr ValSer Phe 325 330 335 cga gac ctg ggc tgg cag gac tgg atc atc gcg cct gaaggc tac gcc 1113 Arg Asp Leu Gly Trp Gln Asp Trp Ile Ile Ala Pro Glu GlyTyr Ala 340 345 350 355 gcc tac tac tgt gag ggg gag tgt gcc ttc cct ctgaac tcc tac atg 1161 Ala Tyr Tyr Cys Glu Gly Glu Cys Ala Phe Pro Leu AsnSer Tyr Met 360 365 370 aac gcc acc aac cac gcc atc gtg cag acg ctg gtccac ttc atc aac 1209 Asn Ala Thr Asn His Ala Ile Val Gln Thr Leu Val HisPhe Ile Asn 375 380 385 ccg gaa acg gtg ccc aag ccc tgc tgt gcg ccc acgcag ctc aat gcc 1257 Pro Glu Thr Val Pro Lys Pro Cys Cys Ala Pro Thr GlnLeu Asn Ala 390 395 400 atc tcc gtc ctc tac ttc gat gac agc tcc aac gtcatc ctg aag aaa 1305 Ile Ser Val Leu Tyr Phe Asp Asp Ser Ser Asn Val IleLeu Lys Lys 405 410 415 tac aga aac atg gtg gtc cgg gcc tgt ggc tgc cactagctcctcc 1351 Tyr Arg Asn Met Val Val Arg Ala Cys Gly Cys His 420 425430 gagaattcag accctttggg gccaagtttt tctggatcct ccattgctcg ccttggccag1411 gaaccagcag accaactgcc ttttgtgaga ccttcccctc cctatcccca actttaaagg1471 tgtgagagta ttaggaaaca tgagcagcat atggcttttg atcagttttt cagtggcagc1531 atccaatgaa caagatccta caagctgtgc aggcaaaacc tagcaggaaa aaaaaacaac1591 gcataaagaa aaatggccgg gccaggtcat tggctgggaa gtctcagcca tgcacggact1651 cgtttccaga ggtaattatg agcgcctacc agccaggcca cccagccgtg ggaggaaggg1711 ggcgtggcaa ggggtgggca cattggtgtc tgtgcgaaag gaaaattgac ccggaagttc1771 ctgtaataaa tgtcacaata aaacgaatga atgaaaaaaa aaaaaaaaaa a 1822 431amino acids amino acid linear protein not provided 2 Met His Val Arg SerLeu Arg Ala Ala Ala Pro His Ser Phe Val Ala 1 5 10 15 Leu Trp Ala ProLeu Phe Leu Leu Arg Ser Ala Leu Ala Asp Phe Ser 20 25 30 Leu Asp Asn GluVal His Ser Ser Phe Ile His Arg Arg Leu Arg Ser 35 40 45 Gln Glu Arg ArgGlu Met Gln Arg Glu Ile Leu Ser Ile Leu Gly Leu 50 55 60 Pro His Arg ProArg Pro His Leu Gln Gly Lys His Asn Ser Ala Pro 65 70 75 80 Met Phe MetLeu Asp Leu Tyr Asn Ala Met Ala Val Glu Glu Gly Gly 85 90 95 Gly Pro GlyGly Gln Gly Phe Ser Tyr Pro Tyr Lys Ala Val Phe Ser 100 105 110 Thr GlnGly Pro Pro Leu Ala Ser Leu Gln Asp Ser His Phe Leu Thr 115 120 125 AspAla Asp Met Val Met Ser Phe Val Asn Leu Val Glu His Asp Lys 130 135 140Glu Phe Phe His Pro Arg Tyr His His Arg Glu Phe Arg Phe Asp Leu 145 150155 160 Ser Lys Ile Pro Glu Gly Glu Ala Val Thr Ala Ala Glu Phe Arg Ile165 170 175 Tyr Lys Asp Tyr Ile Arg Glu Arg Phe Asp Asn Glu Thr Phe ArgIle 180 185 190 Ser Val Tyr Gln Val Leu Gln Glu His Leu Gly Arg Glu SerAsp Leu 195 200 205 Phe Leu Leu Asp Ser Arg Thr Leu Trp Ala Ser Glu GluGly Trp Leu 210 215 220 Val Phe Asp Ile Thr Ala Thr Ser Asn His Trp ValVal Asn Pro Arg 225 230 235 240 His Asn Leu Gly Leu Gln Leu Ser Val GluThr Leu Asp Gly Gln Ser 245 250 255 Ile Asn Pro Lys Leu Ala Gly Leu IleGly Arg His Gly Pro Gln Asn 260 265 270 Lys Gln Pro Phe Met Val Ala PhePhe Lys Ala Thr Glu Val His Phe 275 280 285 Arg Ser Ile Arg Ser Thr GlySer Lys Gln Arg Ser Gln Asn Arg Ser 290 295 300 Lys Thr Pro Lys Asn GlnGlu Ala Leu Arg Met Ala Asn Val Ala Glu 305 310 315 320 Asn Ser Ser SerAsp Gln Arg Gln Ala Cys Lys Lys His Glu Leu Tyr 325 330 335 Val Ser PheArg Asp Leu Gly Trp Gln Asp Trp Ile Ile Ala Pro Glu 340 345 350 Gly TyrAla Ala Tyr Tyr Cys Glu Gly Glu Cys Ala Phe Pro Leu Asn 355 360 365 SerTyr Met Asn Ala Thr Asn His Ala Ile Val Gln Thr Leu Val His 370 375 380Phe Ile Asn Pro Glu Thr Val Pro Lys Pro Cys Cys Ala Pro Thr Gln 385 390395 400 Leu Asn Ala Ile Ser Val Leu Tyr Phe Asp Asp Ser Ser Asn Val Ile405 410 415 Leu Lys Lys Tyr Arg Asn Met Val Val Arg Ala Cys Gly Cys His420 425 430 102 amino acids amino acid linear protein not providedProtein 1..102 /label= OPX /note= “Wherein each Xaa is independentlyselected from a group of one or more specified amino acids as defined inthe specification” 3 Cys Xaa Xaa His Glu Leu Tyr Val Xaa Phe Xaa Asp LeuGly Trp Xaa 1 5 10 15 Asp Trp Xaa Ile Ala Pro Xaa Gly Tyr Xaa Ala TyrTyr Cys Glu Gly 20 25 30 Glu Cys Xaa Phe Pro Leu Xaa Ser Xaa Met Asn AlaThr Asn His Ala 35 40 45 Ile Xaa Gln Xaa Leu Val His Xaa Xaa Xaa Pro XaaXaa Val Pro Lys 50 55 60 Xaa Cys Cys Ala Pro Thr Xaa Leu Xaa Ala Xaa SerVal Leu Tyr Xaa 65 70 75 80 Asp Xaa Ser Xaa Asn Val Xaa Leu Xaa Lys XaaArg Asn Met Val Val 85 90 95 Xaa Ala Cys Gly Cys His 100 97 amino acidsamino acid linear protein not provided Protein 1..97 /label= GenericSequence 7 /note= “Wherein each Xaa is independently selected from agroup of one or more specified amino acids as defined in thespecification” 4 Leu Xaa Xaa Xaa Phe Xaa Xaa Xaa Gly Trp Xaa Xaa Xaa XaaXaa Xaa 1 5 10 15 Pro Xaa Xaa Xaa Xaa Ala Xaa Tyr Cys Xaa Gly Xaa CysXaa Xaa Pro 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn His Ala Xaa XaaXaa Xaa Xaa 35 40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa CysCys Xaa Pro 50 55 60 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa XaaXaa Xaa Xaa 65 70 75 80 Val Xaa Leu Xaa Xaa Xaa Xaa Xaa Met Xaa Val XaaXaa Cys Xaa Cys 85 90 95 Xaa 102 amino acids amino acid linear proteinnot provided Protein 1..102 /label= Generic Sequence 8 /note= “Whereineach Xaa is independently selected from a group of one or more specifiedamino acids as defined in the specification” 5 Cys Xaa Xaa Xaa Xaa LeuXaa Xaa Xaa Phe Xaa Xaa Xaa Gly Trp Xaa 1 5 10 15 Xaa Xaa Xaa Xaa XaaPro Xaa Xaa Xaa Xaa Ala Xaa Tyr Cys Xaa Gly 20 25 30 Xaa Cys Xaa Xaa ProXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn His Ala 35 40 45 Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60 Xaa Cys Cys Xaa ProXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa Xaa 65 70 75 80 Xaa Xaa Xaa XaaXaa Val Xaa Leu Xaa Xaa Xaa Xaa Xaa Met Xaa Val 85 90 95 Xaa Xaa Cys XaaCys Xaa 100 5 amino acids amino acid linear protein not provided Protein1..5 /label= Consensus Sequence /note= “Wherein each Xaa isindependently selected from a group of one or more specified amino acidsas defined in the specification” 6 Cys Xaa Xaa Xaa Xaa 1 5 97 aminoacids amino acid linear protein not provided Protein 1..97 /label=Generic Sequence 10 /note= “Wherein each Xaa is independently selectedfrom a group of one or more specified amino acids as defined in thespecification” 7 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa 1 5 10 15 Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Gly Xaa CysXaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa 35 40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaCys Xaa Pro 50 55 60 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa XaaXaa Xaa Xaa 65 70 75 80 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Cys Xaa Cys 85 90 95 Xaa 102 amino acids amino acid linear proteinnot provided Protein 1..102 /label= Generic Sequence 9 /note= “Whereineach Xaa is independently selected from a group of one or more specifiedamino acids as defined in the specification” 8 Cys Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa XaaPro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Gly 20 25 30 Xaa Cys Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45 Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60 Xaa Xaa Cys Xaa ProXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa Xaa 65 70 75 80 Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 85 90 95 Xaa Xaa Cys XaaCys Xaa 100

What is claimed is:
 1. A method for inducing bone formation in a mammalsufficient to fill a defect locus defining a void, the method comprisingthe step of providing directly to said locus defining said void anosteogenic device comprising an osteogenic protein capable of inducingrepair or regeneration of endochondral bone, intramembranous bone,cartilage, chondral or osteochondral defects, dispersed in abiocompatible, non-rigid amorphous carrier having no defined surfaces;with the proviso that said osteogenic protein is not TGF-β and that saidcarrier is not atelopeptide collagen.
 2. The method of claim 1 whereinthe volume of said device provided to said defect locus is insufficientto fill said void.
 3. The method of claim 1 wherein said device lacksscaffolding structure.
 4. The method of claim 1 wherein said defectlocus defines a volume incapable of endogenous repair.
 5. The method ofclaim 1 wherein said bone formation is endochondral bone formation. 6.The method of claim 1 wherein said bone formation is intramembranousbone formation.
 7. The method of claim 1 wherein said carrier comprisesa gel.
 8. The method of claim 1 wherein said carrier comprises anaqueous solution.
 9. The method of claim 1 wherein said carrier isselected from the group consisting of: alkylcelluloses; poloxamers;gelatins; polyethylene glycols (PEG); dextrins; and vegetable oils. 10.The method of claim 1 wherein said carrier is selected from the groupconsisting of: carboxymethylcellulose; mannitol; PEG 3350; poloxamer407; and sesame oil.
 11. The method of claim 1 wherein said osteogenicprotein is selected from the group consisting of: OP1; OP2; OP3; BMP2;BMP3; BMP4; BMP5; BMP6; BMP9; BMP-10; BMP-11; BMP-12 ; BMP-15; BMP-3b;DPP; Vg1; Vgr; 60A protein; GDF-1; GDF-3; GDF-5; GDF-6; GDF-7; GDF-8;GDF-9; GDF-10; GDF-11; and amino acid sequence variants thereof.
 12. Themethod of claim 1 wherein said osteogenic protein is selected from thegroup consisting of: OP1; OP2; BMP2; BMP4; BMP5; BMP6; and amino acidsequence variants thereof.
 13. The method of claim 1 wherein saidosteogenic protein is a morphogen, said morphogen comprising an aminoacid sequence having at least 70% homology within the C-terminal 102-106amino acids, including the conserved seven cysteine domain, of humanOP1, said osteogenic protein being capable of inducing repair orregeneration of endochondral bone, intramembranous bone, cartilage,chondral or osteochondral defects.
 14. The method of claim 1 whereinsaid osteogenic protein is OP1.
 15. The method of claim 1 wherein saidosteogenic protein is mature OP1 solubilized in a saline solution.
 16. Amethod for inducing bone formation in a mammal sufficient to fill adefect locus defining a void, the method comprising the step ofproviding to said locus substantially pure osteogenic protein free of acarrier or scaffolding structure.
 17. A method for enhancing thequantity or quality of callus formation at an osteogenic defect locus ina mammal, the method comprising the step of administering directly tosaid locous an osteogenic device comprising an osteogenic proteincapable of inducing repair or regeneration of endochondral bone,intramembranous bone, cartilage, chondral or osteochondral defects,dispersed in a biocompatible, non-rigid amorphous carrier having nodefined surfaces; with the proviso that said osteogenic protein is notTGF-β and that said carrier is not atelopeptide collagen.
 18. The methodof claim 17 wherein said osteogenic protein is a morphogen, saidmorphogen comprising an amino acid sequence having at least 70% homologywithin the C-terminal 102-106 amino acids, including the conserved sevencysteine domain, of human OP1, said osteogenic protein being capable ofinducing repair or regeneration of endochondral bone, intramembranousbone, cartilage, chondral or osteochondral defects.
 19. The method ofclaim 17 wherein said osteogenic protein is OP1.
 20. The method of claim17 wherein said osteogenic protein comprises an amino acid sequencedefined by OPX (Seq. ID No. 3); Generic Sequence 7 (Seq. ID No. 4),Generic Sequence 8 (Seq. ID No. 5); Generic Sequence 10 (Seq. ID No. 7);or Generic Sequence 9 (Seq. ID No. 8).
 21. A method of accelerating boneformation, the method comprising the step of providing directly to adefect locus an osteogenic device comprising an osteogenic proteincapable of inducing repair or regeneration of endochondral bone,intramembranous bone, cartilage, chondral or osteochondral defects,dispersed in a biocompatible, non-rigid amorphous carrier having nodefined surfaces; with the proviso that said osteogenic protein is notTGF-β and that said carrier is not atelopeptide collagen.
 22. A methodof inducing endogenous matrix formation, the method comprising the stepof providing directly to a defect locus an osteogenic device comprisingan osteogenic protein capable of inducing repair or regeneration ofendochondral bone, intramembranous bone, cartilage, chondral orosteochondral defects, dispersed in a biocompatible, non-rigid amorphouscarrier having no defined surfaces; with the proviso that saidosteogenic protein is not TGF-β and that said carrier is notatelopeptide collagen.
 23. A method of repairing a bone defect, chondraldefect or osteochondral defect, said method comprising the step ofadministering directly to a defect a matrix-free osteogenic devicecomprising an osteogenic protein capable of inducing repair orregeneration of endochondral bone, intramembranous bone, cartilage,chondral or osteochondral defects, wherein administering said devicepost-injury is delayed; with the proviso that said osteogenic protein isnot TGF-β and that said carrier is not atelopeptide collagen.
 24. Themethod of claim 23 wherein said administering step is delayed at least 6hours post-injury.
 25. A method of repairing a chondral defect, saidmethod comprising the step of administering directly to a chondraldefect an osteogenic device comprising osteogenic protein and aglycosaminoglycan carrier; with the proviso that said osteogenic proteinis not TGF-β.